Simian virus 40 (SV40) large T antigen binds to host cell DNA polymerase α and the T-antigen-DNA polymerase α complex is implicated in the initiation of viral DNA replication. We have examined various SV40 T-antigen mutants to test the correspondence between viral DNA replication and T-antigen-DNA polymerase α complex formation. The various SV40 T-antigen mutants were used to either infect or transfect African green monkey kidney cell line CV-1, and at different time intervals we measured the production of T-antigen and host cell DNA polymerase α by radioimmunoassay, complex formation by a sandwich radioimmunoassay and the amount of viral DNA synthesis by dot-blot hybridization analysis. There was a good correlation between complex formation and viral DNA synthesis in lytic mutants of SV40. Poor complex formation and correspondingly lower DNA synthesis were observed in the non-viable mutants of SV40, even though significant amounts of T-antigen and DNA polymerase α were present. Our results substantiate the earlier findings of T-antigen-DNA polymerase α complex formation and establish the need for formation of this complex in promoting viral DNA synthesis.