The dinucleotide CA/TG found at the termini of transposable phage Mu occurs also at the termini of a large class of transposable elements, including HIV, all retroviruses and many retrotransposons. It was shown recently that mutations of this sequence block transpososome assembly, that A/T is more critical for activity than C/G, and that the hierarchy of reactivity of mutant termini follows closely the reported hierarchy of flexibility of their dinucleotide steps. In order to test the hypothesis that the terminal dinucleotide plays an essential structural role during "open termini" formation accompanying assembly, we have examined the activity of substrates carrying 100 different pairs of mismatched termini. Consistent with the flexibility hypothesis, we find that mismatched substrates are extremely efficient at assembly. A wild-type T residue on the bottom strand is essential for stable assembly, but the identity of the dinucleotide on the top strand is irrelevant for transposition chemistry. In addition, we have found a new rule for suppression of terminal defects by MuB protein, as well as a role for metal ions in DNA opening at the termini.