Yeast DNA polymerase delta (Poldelta) consists of three subunits encoded by the POL3, POL31, and POL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1-10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30-fold overproduction of Poldelta, which was identical in enzymatic properties to Poldelta isolated from a wild-type yeast strain. Whereas overproduction of POL3 together with POL32 did not lead to an identifiable Pol3p.Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 and POL31. This two-subunit complex, designated Poldelta*, is structurally and functionally analogous to mammalian Poldelta. The properties of Poldelta* and Poldelta were compared. A gel filtration analysis showed that Poldelta* is a heterodimer (Pol3p.Pol31p) and Poldelta a dimer of a heterotrimer, (Pol3p.Pol31p.Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Poldelta* showed a processivity of 2-3 on poly(dA). oligo(dT) compared with 5-10 for Poldelta. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Poldelta* on a natural DNA template was dependent on PCNA and on replication factor C. However, Poldelta*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Poldelta was achieved upon addition of Pol32p to Poldelta*.