The sister chromatid cohesion factor Chl12 shares amino acid sequence similarity with RFC1, the largest subunit of replication factor C (RFC), and forms a clamp loader complex in association with the RFC small subunits RFCs2-5. It has been shown that the human Chl12-RFC complex, reconstituted with a baculovirus expression system, specifically interacts with human proliferating cell nuclear antigen (PCNA). The purified Chl12-RFC complex is structurally indistinguishable from RFC, as shown by electron microscopy, and it exhibits DNA-stimulated ATPase activity that is further enhanced by PCNA, and by DNA binding activity on specific primer/template DNA structures. Furthermore, the complex loads PCNA onto a circular DNA substrate, and stimulates DNA polymerase delta DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC.