We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase a and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.The REV3 gene of bakers' yeast, Saccharomyces cerevisiae, is concerned with a recovery process in which DNA damage causes mutations within the nuclear genome. It was identified by isolation of strains displaying reduced frequencies of UV mutagenesis (28), and, compared with others selected by such a criterion (25,26,28,32,37), strains carrying rev3 mutations exhibit one of the most extreme and general deficiencies in this respect. Relative to wild type, rev3 mutants display a 96% reduction in forward mutation to auxotrophy induced by UV (31) and also display defects in UV-induced reversion of broad range of base substitution and frameshift test alleles (24,27,28). The rev3 mutant is also deficient in mutagenesis by 4-nitroquinoline-1-oxide (41) and gamma rays (34), though response to ethyl methanesulfonate and nitrous acid is normal (31, 41). Spontaneous mutation is reduced to about one-fifth of normal in rev3 mutants, suggesting that the REV3-dependent recovery process may act on spontaneously arising damage (42). It does not, however, appear to be concerned with damage in the mitochondrial genome: UV-induced mutagenesis in mitochondrial DNA is normal in rev3 mutants (40, 45). These observations suggest that the REV3 gene product may function in translesion synthesis, that is in replication on mutagen-damaged templates, though they do not point to any specific role. The REV] gene (28), which has a mutant phenotype similar to that of REV3 and was identified in the same way, has recently been shown to encode a predicted protein of Mr 112,239 with sequence similarity to the Escherichia coli umuC gene (22). The umuDC genes of E. coli also have a mutant phenotype similar to that of REV3 and encode 16,000-and 45,000-Mr proteins that are thought to enhance the processivity of DNA polymerase (1,11