The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.