Transposition of Tn5 and IS50 requires the specific binding of transposase (Tnp) to the end inverted repeats, the outside end (OE) and the inside end (IE). OE and IE have 12 identical base-pairs and seven non-identical base-pairs. Previously we described the isolation of a Tnp mutant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp. EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition. Here we report that base-pairs 10, 11 and 12 of the OE are critical for the specific recognition by EK54 Tnp. These three adjacent base-pairs act cooperatively; all three must be present in order for EK54 Tnp to interact very favorably with the end DNA. The existence of only one or two of these three base-pairs decreases binding of EK54 Tnp. The combined use of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE base-pairs 10, 11 and 12 gives rise to an extraordinarily high transposition frequency. Just as the Tnp is a multifunctional protein, the nucleotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding. Furthermore, the fact that we were able to isolate end sequence variants that transpose at a higher frequency than the natural ends (OE and IE) with wt Tnp reveals yet another way in which the wt transposition frequency is depressed, i.e. by keeping the end sequences suboptimal.