Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage f1 replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on f1 viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of f1 viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on f1 complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the f1 replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for f1 DNA replication in vivo proposed by Horiuchi and Zinder.