Simian virus 40 large T antigen is the only viral protein required for SV40 DNA synthesis in vivo and in vitro. This complex protein recruits the cellular DNA replication apparatus to the SV40 origin and provides a good model for the initiation of cellular DNA replication. The interaction between SV40 large T antigen (TAg) and DNA polymerase alpha has been shown previously to be inhibited by murine p53, the nuclear protein product of a cellular anti-oncogene. The murine p53 protein will inhibit SV40 replication both in vivo and in vitro. Using monoclonal antibodies to TAg, p53, and polymerase alpha, we developed immunoassays to measure the complexes formed between TAg and polymerase alpha and between TAg and p53. The assays allowed us to detect the TAg-polymerase alpha and TAg-p53 complexes in lytically infected and transformed cells. The amount of TAg complexed to p53 was far lower in infected cells than in transformed cells. We used a large range of monoclonal antibodies to different sites on T antigen and found that antibodies that inhibited the formation of the TAg-polymerase alpha complex also inhibited the formation of the TAg-p53 complex. Finally, we found that the tsA58 and 5080 point mutations in TAg, previously shown to inhibit the binding of TAg to p53, also inhibit its binding to polymerase alpha. Together these results emphasize the specificity and functional importance of the TAg-polymerase alpha complex. The disruption of this interaction by the cellular anti-oncogene p53 provides an interesting model for the normal action of p53 and the effects of its removal on the regulation of cellular DNA synthesis.