Proliferating cell nuclear antigen (PCNA) is required for mismatch repair (MMR) and has been shown to interact with complexes containing Msh2p or MLH1 (refs 1-4). PCNA has been implicated to act in MMR before and during the DNA synthesis step, although the biochemical basis for the role of PCNA early in MMR is unclear. Here we observe an interaction between PCNA and Msh2p-Msh6p mediated by a specific PCNA-binding site present in Msh6p. An msh6 mutation that eliminated the PCNA-binding site caused a mutator phenotype and a defect in the interaction with PCNA. The association of PCNA with Msh2p-Msh6p stimulated the preferential binding of Msh2p-Msh6p to DNA containing mispaired bases. Mutant PCNA proteins encoded by MMR-defective pol30 alleles were defective for interaction with Msh2p-Msh6p and for stimulation of mispair binding by Msh2p-Msh6p. Our results suggest that PCNA functions directly in mispair recognition and that mispair recognition requires a higher-order complex containing proteins in addition to Msh2p-Msh6p.