The yeast Saccharomyces cerevisiae encodes four proteins, Msh1, Msh2, Msh3, Msh4, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. MutS has been shown to recognize base pair mismatches in DNA in vitro. Previous studies have suggested that Msh2 is the major mismatch recognition protein in yeast. In this study, the 109-kD Msh2 polypeptide was overexpressed and purified to analyze its DNA-binding properties. This analysis demonstrated that Msh2 can bind selectively to duplex oligonucleotide substrates containing a G/T mismatch, 1- to 14-nucleotide insertion mismatches, and palindromic (12- to 14-nucleotide) insertion mismatches. A general trend was that the affinity of Msh2 for substrate was proportional to the size of the insertion mispair present (+14 PAL, +12 PAL > +14 > +8 > GT, +6, +4, +2, +1). Kinetic studies indicated that the specificity of Msh2 to mismatch substrates was a function of its ability to form stable complexes with mispair-containing duplex DNAs. These complexes decayed more slowly than Msh2 complexes formed with homoduplex DNA.