This study was initiated with the objective of studying various aspects of the physical and chemical structure of otoliths of humpback chub from the Grand Canyon of Arizona, and primarily from the Little Colorado River (LCR). These studies were to shed light on the utility of otolith studies for improving knowledge of the life history of this endangered species, and to evaluate the potential application of these techniques to questions posed by resource managers. Whole fish specimens, skeletons, or preserved heads of specimens were provided to the author by a diversity of Arizona Game and Fish field crews who collected from 1989 through 1993. The author, with assistance of Dr. Ed Brothers, extracted, prepared and examined otoliths. Data analysis and reporting was the sole responsibility of the author. Specifically, the study was to obtain age estimates (years of age) from otoliths of 50 selected skeletonized adult specimens of Gila cypha collected from the Grand Canyon by Arizona Game and Fish Department in 1989 and 1990. It was also to obtain age estimates (days of age) for 100 selected young-of-the-year (y.o.y.) Gila cypha collected during the same and subsequent years. Age estimates for y.o.y. were predicated on the assumption that increments counted in the otoliths were deposited daily, and that increment counts could thus be translated to days of life since the date of first increment formation (generally within the first few days following spawning). Since that hypothesis had not been specifically tested in this species, the study also was to test the hypothesis that increments form on a daily basis, both in the field and in hatchery experiments. Since at least some humpback chub appear to move across a typically strong thermal gradient at the interface of mainstem Colorado River (MCR) waters and the discharge of the LCR, which is generally much warmer than MCR, it was hypothesized that this transition might lead to the formation of marks, both physical and chemical, in otoliths and that these marks might be used to reconstruct individual life histories with respect to timing of this inter-river movement. Though studies conducted since initiation of the present study \9017\ have recently made significant contributions toward documentation of movements of adult humpback chub in the mainstem Colorado, still very little is known of movements of y.o.y. It had been hypothesized that if swept out of the LCR into the mainstem Colorado, the transition might be lethal or have other deleterious impacts on y.o.y. survival and growth. A mark in otoliths that unambiguously conveyed information about extent and timing of movements across this inter-river interface, could thus be valuable in furthering understanding of population dynamics and movements. It was thus proposed to search for such marks in otoliths and to conduct experiments to study the effects of temperature changes on otolith structure. The original study design also called for an analysis of the feasibility of determining annual growth period duration from otoliths of post young-of-the-year individuals of Gila cypha for all growth periods throughout the life of specimens. At the time of study design, there was considerable discussion and application of chemical analyses of otoliths in the literature of fishery management and stock identification. Studies at this time indicated considerable promise for the techniques, and likely applicability to reconstruction of detailed individual life histories of humpback chub. It was hypothesized that individuals that moved across the MCR-LCR temperature and water quality gradient would deposit a chemical/structural signal in their otoliths that reflected this transtion from one river to the other. Since the temporal structure of otolith deposition and specimen birth date could be recovered from the otoliths as well, the absolute date of the movement event, and fish size at the time, might be accurately recoverable as well. It was therefore proposed to carry out analyses of micro-spatial (=chronological) variation in elemental composition in otoliths of 20 selected individual Gila cypha specimens from the Grand Canyon for evaluation of the utility of such techniques for reconstruction of movement history of individuals. In addition it was hoped to compare total elemental composition among otoliths of 5 selected individual specimens of young-of-the-year Gila cypha captured in the Little Colorado River, otoliths of 5 hatchery-reared young-of-the-year Gila cypha, and otoliths of 5 selected Gila cypha suspected or known to have moved between the Little Colorado River and mainstem Colorado River in the Grand Canyon as a means of investigating the effect of these diverse environments on otolith composition. An accidental spill of isotopes into the LCR drainage \9018\ was thought to potentially provide isotopic signatures in otoliths. If some of the isotopes characteristic of the spill were found in otoliths, their presence might serve as an unambiguous marker indicating time spent in the LCR. It was therefore proposed to determine the isotopic composition of a subsamplc of the same (or comparable) specimens used for microchemical composition studies. Since otolith isotopic composition had been indicated in other studies tu he highly correlated with ambient temperature, isotopic compositional changes during the temporal sequence of otolith deposition thus might also reflect inter-river transitions. Specimens from experiments designed to determine the effects of ambient temperature on otolith increment deposition were therefore to be examined for isotopic composition as well in an attempt to better understand the effect of temperature on isotopic composition of otoliths. In the course of these studies a bibliography of literature relevant to methods and problems of estimating age and growth of Gila cypha and chemical composition of otoliths as related to application of otolith chemistry to reconstruction of the environmental history of individuals was compiled and is provided with this report. Though this bibliography can hardly be claimed to he comprehensive since the literature in this field has become very extensive, it should serve as a starting point for future researchers interested in otolith studies. Finally, the appendices of this report provide an inventory of all specimens of Gila cypha from the Grand Canyon used (and not used) in this study, and the earlier interim report on early results from this study (less the bibliography, which has been updated in this report). Some questions answered in that report, such as comparisons of ageing techniques using the asteriscus and opercle, are not reiterated here, and the figures provided there amply illustrate all otolith structural features and variations discussed in this report.