The Escherichia coli mutS gene product is involved in mismatch correction in this organism. We have purified a biologically active form of the 97,000 Mr protein to near homogeneity from an overproducing strain. Enzymatic and chemical protection ("footprinting") experiments have demonstrated that mutS-encoded protein specifically binds to DNA regions containing a single base-pair mismatch. The protein displayed variable affinity for the limited set of mismatches tested (G-T greater than G-A approximately equal to A-C greater than T-C).