Replication through (6-4)TT and G-AAF lesions was compared in Saccharomyces cerevisiae strains proficient and deficient for the RAD30-encoded DNA polymerase eta (Pol eta). In the RAD30 strain, the (6-4)TT lesion is replicated both inaccurately and accurately 60 and 40% of the time, respectively. Surprisingly, in a rad30 Delta strain, the level of mutagenic bypass is essentially suppressed, while error-free bypass remains unchanged. Therefore, Pol eta is responsible for mutagenic replication through the (6-4)TT photoproduct, while another polymerase mediates its error-free bypass. Deletion of the RAD30 gene also reduces the levels of both accurate and inaccurate bypass of AAF lesions within two different sequence contexts up to 8-fold. These data show that, in contrast to the accurate bypass by Pol eta of TT cyclobutane dimers, it is responsible for the mutagenic bypass of other lesions. In conclusion, this paper shows that, in yeast, translesion synthesis involves the combined action of several polymerases.