The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase for processive DNA synthesis has so far not been clearly identified. We show here that the putative third subunit of human DNA polymerase , p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C-terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase activity and for binding of the cell cycle inhibitor p21^Cip1. In accordance with this, a peptide containing the PCNA-binding domain of p21^Cip1inhibited p66 binding to PCNA as well as the activity of native three-subunit DNA polymerase . Furthermore, pull-down assays showed that DNA polymerase requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase displayed PCNA-dependent DNA replication that could be inhibited by a peptide containing the PCNA-binding domain of p21^Cip1. Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and the catalytic subunit (125 kDa) of DNA polymerase within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle dependent regulation. Taken together, these results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase .