Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast. RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa. None of the subunits are related through proteolysis or differential phosphorylation. The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA. This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA). In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well. Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule. DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C. RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA. Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus. Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta. These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.