The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol d) holoenzyme on DNA templates. Previous studies have shown that S. cerevisiae and human PCNA do not complement each other using in vitro or in vivo assays. Since both proteins exist as trimers, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol d holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2,CH3, CH4 and CH5, were prepared by swapping corresponding regions between the two proteins.In solution, the mutants and hybrids assembled into trimers, albeit to different extents. These PCNA variants were tested for their ability to support pol d catalysed DNA synthesis and in vitro replication of M13 and SV40 as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the previously defined sites in the inter-domain connecting loop and C-terminus, an additional site in the N-terminus is required for PCNA to interact with pol d. Significant RF-C stimulation with the hybrid CH2, which was predominantly a dimer in solution,suggested that trimeric PCNA may not be a pre-requisite for RF-C mediated clamping. PCNA mutant HU3 and hybrids CH3 and CH5 which stimulated pol d and RF-C were deficient in M13 and SV40 DNA replication assays indicating that PCNA-induced stimulation of pol d and RF-C mediated loading is not sufficient for co-ordinated DNA synthesis at a replication fork. The data are consistent with the view that participation of other proteins may be necessary in order to assemble a functional pol d holoenzyme complex at a replication fork.