It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most to the inhibition of pol delta holoenzyme activity. p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA. Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.