Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10(7) M-1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.