The human MutSalpha protein, a heterodimer between MSH2 and MSH6, initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2 G674A or Msh6 T1217D mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6, respectively, develop cancer and have reduced lifespan due to loss of the MMR pathway. Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSa proteins, MSH2 G674A ‐MSH6 wt and MSH2 wt ‐MSH6 T1219D , are profiled in a variety of functional assays and, as expected, fail to support MMR in vitro , although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2 wt ‐MSH6 T1219D fails to couple nucleotide binding and mismatch recognition; whereas, MSH2 G674A ‐MSH6 wt has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Our findings highlight differences in the molecular defects of the G674A and T1219D mutant MutSalpha proteins and provide a basis for thinking about how they might mediate the apoptotic response to certain DNA damaging agents.