P1 prophage replication during the Escherichia coli division cycle has been analyzed by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive prophage DNA. P1 prophage replicates during a restricted portion of the bacterial division cycle, like the minichromosome, but at a time during the division cycle different than the time at which the minichromosome replicates in the same cell. A high-copy mini-R6K plasmid present in the same cell replicates throughout the division cycle. Over a wide range of growth rates, the P1 prophage replicates approximately one-half generation after the minichromosome replicates. Thus, the mechanisms underlying P1 replication are similar to those for the F plasmid and the chromosome. Replication occurs when some property related to cell size or cell mass reaches a constant value per origin.