Insertion sequence 50 (IS50) transposition utilizes a 19-base-pair "outside" end and a 19-base-pair "inside" end in inverted orientation relative to each other, whereas transposon 5 (Tn5) transposition utilizes two inverted outside ends. The frequency of transposition events that involve an inside end is regulated 1000-fold by the host dam methylase system. The end sequence requirements for transposition and its regulation by dam methylase were analyzed in Escherichia coli by generating random single base pair mutations in either an IS50 inside end or outside end placed in inverted orientation with respect to an unmutagenized outside end. The mutations were then isolated, assayed for transposition phenotype, and sequenced. Mutations were isolated at 15 of the 19 sites in the outside end. All of these mutations except those at position 4 decreased transposition. Mutations at position 4 (which is the only nonidentical base pair in a region of homology between the outside and inside ends) had no effect on transposition. Mutations were isolated at 11 of the 19 sites in the inside end. All of these mutations, including one at position 4, decreased transposition in dam- cells. Mutations at position 10 (within a dam recognition sequence) and 2 (not within a dam recognition sequence) reduced the magnitude of dam regulation. A mutation within a dam recognition sequence adjacent to the required 19 base pairs of the inside end did not reduce the magnitude of dam regulation.