We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 T antigen to mediate the replication of a plasmid bearing the viral origin (ori-DNA) in vitro. In contrast, polyoma ori-DNA replication in vitro was unaffected by p53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 ori-DNA replication. Thus, p53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of ori-DNA replication. In contrast to the nononcogenic wild-type murine p53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell.