The bacterial transposon Tn7 is distinguished by its capacity for high-frequency transposition to a specific site in the Escherichia coli chromosome. tnsB is one of the five Tn7-encoded transposition genes. We have identified in vitro a tnsB-dependent DNA binding activity that interacts specifically with cis-acting transposition sequences at the Tn7 termini. Although the left and right termini of Tn7 are structurally distinct, each end contains several copies of a closely homologous 22-base-pair sequence. We present results indicating that this 22-base-pair repeat sequence is recognized by the tnsB-dependent binding activity.