By assaying transposition of Tn5 from lambda b221 cI857 rex::Tn5 (Berg 1977) in polA-proficient and deficient cells, both the polymerase activity and 5' to 3' exonuclease activity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even PolA-deficient cells still possessing a residual activity. One PolA mutant was found among more than 50 transposition deficient (tnp) mutants isolated by the use of lambda b221 cI857 rex::Tn5.