Significance MutLα (MLH1-PMS2 heterodimer) is an endonuclease that acts during an early step of eukaryotic mismatch repair. We show that human MutLα endonuclease copurifies with two equivalents of bound zinc, at least one of which resides within the endonuclease active site. We also show that cadmium, a known inhibitor of zinc-dependent enzymes and a potent mutagen and carcinogen, is a high-affinity inhibitor of MutLα endonuclease and that exogenous MutLα significantly reverses the mismatch repair defect in cadmium-treated human cell nuclear extract or nuclear extract prepared from cadmium-treated cells. Because the mutagenic action of cadmium is largely due to the selective inhibition of mismatch repair, these findings suggest that MutLα is a primary cadmium target for mutagenesis and presumably, carcinogenesis as well.