The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and Cll cells (CV-1 cell lines transformed with UVirradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC1l. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys --Arg at amino acid 516. pC1l has two mutations, one predicting a change of Pro -* Ser at residue 522, and another predicting a Pro --Arg change at amino acid 549. The two Cll mutations were separated from each other to form two distinct viral genomes in pCllA and pC11B. pC2, pCll, pC11A, and pCllB are able to transform both primary and established rodent cell cultures. The Cll and C1lA T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.The multifunctional large T antigen of simian virus 40 (SV40) is essential in both the lytic cycle of the virus and the oncogenic transformation of cells by SV40. T antigen, encoded by the SV40 A gene, is expressed early in the lytic pathway and functions in numerous processes throughout the infectious cycle. It is required for the initiation of viral DNA replication (59), autoregulates early viral RNA synthesis (1, 51, 52, 61), stimulates late viral transcription (30;