The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.