The product of the P gene of bacteriophage lambda was isolated from heat-induced lambda-lysogenic Escherichia coli cells. It was found to bind to DNA, to be devoid of nuclease activity acting on double-stranded lambda DNA and of nicking/closing activity. Initiation of lambda DNA replication promoted by the P-gene product in a complementation assay in vitro was sensitive to rifampicin. Sedimentation analysis of the products and their hybridization to separated lambda DNA strands indicate that lambda DNA was formed in a reaction similar to ring-to-ring replication in vivo. The reaction was symmetric from the beginning, i.e. both lambda DNA strands were copied without delay.