In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.