UV light is a potent mutagen in most living cells, but molecular analysis of its mode of action in animal cells has not been possible because of the high complexity of the cell genome. Therefore, we have used simian virus 40 (SV40) as a biological probe to study the mutagenic effect of UV-irradiation at the molecular level. A thermosensitive SV40 mutant of the large T antigen (tsA58), which is defective at high temperature for initiation of viral DNA replication and for repression of early transcription, was UV-irradiated in vitro, then replicated in monkey kidney cells. Revertants of tsA58 were selected for growth at the nonpermissive temperature, then the reversion sites were mapped by using the marker rescue technique, and precisely localized by DNA sequencing. We report here that all revertants analysed showed one or two base pair substitutions localized in the C-terminal half of the T antigen gene. The original tsA58 mutation was still present in all revertant genomes. In 16 DNAs sequenced, seven reversion sites were found on the large T antigen gene, all of which localized opposite a possible UV-induced pyrimidine-pyrimidine lesion, suggesting targeted mutagenesis. UV-irradiation of the host cell before infection did not change the pattern of reversion sites at the molecular level, although it strongly increased the mutation frequency. These results demonstrate for the first time in animal cells the specificity of UV-induced mutagenesis.