DNA polymerases alpha, delta, and epsilon have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type. The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different. Likewise, the small subunit of DNA polymerase epsilon appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase alpha. HeLa DNA polymerase delta is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase epsilon is quite processive in its absence. Inhibitor and activator spectra of DNA polymerases alpha, delta, and epsilon also distinguish the three enzymes. These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases alpha, delta, and epsilon are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.