Aphidicolin, a specific inhibitor of DNA polymerase a, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (on) and continuation of replication beyond on. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600-to 700-base-pair region centered at on. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 on-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the on region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of on, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase a. Synthesis of pppA/G(pN)64(pdN)21_23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at on first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.Simian virus 40 (SV40) is a small (5.2 kilobases [kb]) DNA virus that replicates in the nuclei of permissive monkey or human cells as a circular chromosome whose nucleosome striicture and histone composition are indistinguishable from that of its host (reviewed in reference 5 and in M. L. DePamphilis and M. K. Bradley, in N. P. Salzman, ed., The Viruses: Polyoma Viruses, in press). With the exception of initiation of viral DNA replication, all steps in the replication and assembly of viral chromosomes are carried out exclusively by cellular components. Initiation of SV40 DNA replication requires a cis-acting sequence that functions as the origin of replication (ori), SV40 large tumor antigen (T-ag), and one or more permissive cell factors. Bidirectional replication then begins at the junction between the strongest DNA-binding site for T-ag and the ori core. Analysis of initiation sites for RNA-primed DNA synthesis in SV40 replicating intermediates led to the hypothesis that initiation of DNA synthesis at ori involves the same mechanism used to synthesize Okazaki...