DNA polymerase delta, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisting of a core enzyme of a 125 kDa catalytic subunit and a smaller 50 kDa subunit. However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication. In this study, the properties of calf thymus pol delta preparations isolated by immunoaffinity chromatography were investigated. It is demonstrated for the first time using highly purified preparations that the pol delta heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to >500000 in size, as determined by FPLC gel filtration. These preparations are associated with polypeptides of ca. 68-70, 34, 32, and 25 kDa. Similar findings were revealed with glycerol gradient ultracentrifugation. The p68 polypeptide was shown to be a PCNA binding protein by overlay methods with biotinylated PCNA. Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039. KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which, like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol delta. These studies provide evidence that p68 is a subunit of pol delta. In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.