We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (delta 17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication.