Initiation of replication of plasmids derived from coliphage lambda in vivo is dependent on transcription at or near the replication origin, ori lambda. However, this transcriptional activation is dispensable for lambda plasmid DNA replication reconstituted in vitro from purified lambda and Escherichia coli proteins. It was proposed previously that histone-like protein HU interferes with the assembly or function of the pre-primosomal complex, and transcription at or near ori lambda abolishes HU-mediated inhibition of lambda DNA replication. We found that during lambda plasmid replication driven by the previously assembled replication complex (in amino acid-starved relA mutants), when the inhibition by HU protein should not be observed, the synthesis of lambda DNA was still dependent on transcriptional activation. Moreover, in hupA hupB double mutants the transcription is necessary for the initiation of lambda plasmid DNA replication perpetuated by the replication complex inherited by one of two daughter copies after a replication round. We conclude that transcriptional activation of ori lambda has an additional role in the initiation of lambda plasmid DNA replication beside the abolition of HU-mediated inhibition.