The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., Camerini-Otero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutSalpha. The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.