Yeast DNA polymerase delta (Poldelta) has three subunits of 125, 58, and 55 kDa. The gene for the 125-kDa catalytic subunit (POL3) has been known for several years. Here we describe the cloning of the genes for the 58- and 55-kDa subunits using peptide sequence analysis and searching of the yeast genome data base. The 58-kDa subunit, encoded by the POL31 gene, shows 23-28% sequence similarity to the 48-kDa subunit of human Poldelta and to S. pombe Cdc1. POL31 is allelic to HYS2 and SDP5. The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base). Very limited sequence similarity was observed between Pol32p and Schizosaccharomyces pombe Cdc27, the functionally analogous subunit in S. pombe Poldelta. The POL32 gene is not essential, but a deletion mutant shows cold sensitivity for growth and is sensitive to hydroxyurea and DNA damaging agents. In addition, lethality was observed when the POL32 deletion mutation was combined with conditional mutations in either the POL3 or POL31 gene. Pol32Delta strains are weak antimutators and are defective for damage-induced mutagenesis. The POL32 gene product binds proliferating cell nuclear antigen. A gel filtration analysis showed that Pol32p is a dimer in solution. When POL31 and POL32 were co-expressed in Escherichia coli, a tetrameric (Pol31p.Pol32p)2 species was detected by gel filtration, indicating that the two subunits form a complex.