THE p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator^1–6. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells^1,3,7. 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) ^5,8. p21 controls CDK activity, thereby affecting cell-cycle control^2–4,6, whereas PCNA functions in both DNA replication^9–12 and repair¹³. Here we use simian virus 40 DNA replication in vitro to show that 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 δ, 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.