Three replicons, R, L and P1dR, have been previously identified in bacteriophage P1, but only the R (or plasmid) replicon has been functionally and structurally characterized. Evidence is provided here that the L-replicon is the principal replicon used for DNA replication during the lytic cycle. The L-replicon (exclusive of its promoter) is shown to be contained within a 1093-base-pair DNA segment that includes a 281-codon open reading frame, designated repL. L-replicon function requires transcription in the direction that should generate translatable repL message. This transcription is normally under the control of the phage c1 repressor, but a deletion that places the functional L-replicon under alternative control can be constructed. The DNA sequence of the replicon and surrounding regions was established. The sequenced region contains the c4 and ant genes and a hitherto unidentified gene, kilA, which is immediately upstream of repL and is controlled by the c1-regulated promoter. The kilA gene was shown to be non-essential for both replication and lytic development whereas the repL gene probably encodes an essential replication protein.