An early SV40 mutant (C6/SV40) was isolated by rescuing the viral DNA integrated into the genome of the C6 cell line which is a CV1 cell line transformed with uv-irradiated SV40. The DNA of C6/SV40 mutant does not replicate detectably in permissive, simian cells, but it transforms them with high efficiency. It also transforms established rat cells with an efficiency equal to that of wild-type DNA, although the phenotype of the mutant-transformed cells is somewhat different from that of the wild-type transformants. Genetic analysis shows that C6/SV40 carries multiple mutations. These mutations were partially segregated into separate viral genomes, C6-1 and C6-2. Nucleotide sequencing showed that the mutations in both C6-1 and C6-2 were transversions (G to T at nucleotide 5074 and A to T at nucleotide 5011 in C6-1, and A to C at nucleotide 4360 in C6-2); all nucleotide numbers are in SV system (J. Tooze (ed.) (1982), “Molecular Biology of Tumor Viruses,” Part 2, “DNA Tumor Viruses,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). The predicted amino acid changes are Met-Ile and Lys-Asn in C6-1 and Asn-Thr in C6-2. In the case of C6-1, these substitutions would occur in the N-terminal portion of large T antigen that is held in common with small t antigen (amino acids 30 and 51). The mutated amino acid in C6-2 resides in the unique portion of large T antigen at position 153. Both C6-1 and C6-2 transform established rat cells as efficiently as wild-type SV40. However, no replication of C6-2 DNA is detectable in permissive CV1 cells, while replication of C6-1 DNA occurs with significantly reduced efficiency. A revertant of C6-1 was isolated and found to have retained the two original mutations and to have acquired a new mutation (G to A at nucleotide 4977), predicting an amino acid change of Glu-Lys at amino acid position 63, which is common to both large and small T antigens.