The cDNA encoding the catalytic polypeptide of human DNA polymerase epsilon was cloned. The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa. A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones. The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA polymerase epsilon. The primary structure comparison with multiple DNA polymerases indicates that human DNA polymerase epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA polymerase II catalytic polypeptide. The proteins are 39% identical. In the region containing known DNA polymerase consensus motifs, the identity is 63%. The expression of the mRNA encoding DNA polymerase epsilon is strongly dependent on cell proliferation.