We have examined interaction parameters, conformation, and functional significance of the human MutSa•PCNA complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 µM in absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSa for a mismatch, and mismatch-bound MutSa binds PCNA. Small angle X-ray scattering studies have established molecular parameters of the complex and are consistent with an elongated conformation in which the two proteins associate in end-to-end fashion in a manner that does not involve an extended unstructured tether as has been proposed for yeast MutSa and PCNA (Shell et al. (2007) Mol. Cell 26, 565-578). MutSa variants lacking the PCNA interaction motif are functional in 3'- or 5'-directed mismatch-provoked excision, but display a partial defect in 5'-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSa PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in mismatch repair.