SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells.