Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition. We report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OE-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA. In addition, two of the Tnp mutants (TP47 and EK54) appear to be change-of-specificity mutants, since they alter the recognition of OE versus IE relative to the wild-type Tnp. EK54 enhances OE recognition but decreases IE recognition. TP47 enhances both OE and IE recognition but with a much greater enhancement for IE than for OE. This change-of-specificity effect of TP47 is observed only when TP47 Tnp is synthesized in cis to the DNA that contains the ends. We propose that Lys54 makes a favorable interaction with an OE-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IE-specific nucleotide pair(s) than it does with the corresponding OE-specific nucleotide pair(s). A model to explain the preference of TP47 Tnp for the IE in cis but not in trans is proposed.