In the yeast Saccharomyces cerevisiae there are five nuclear MutS homologs that act in two distinct processes. MSH2, 3, and 6 function in mismatch repair in both vegetative and meiotic cells, whereas MSH4 and MSH5 act specifically to facilitate crossovers between homologs during meiosis. Coimmunoprecipitation as well as two-hybrid experiments indicate that the Msh4 and Msh5 proteins form a hetero-oligomeric structure similar to what is observed for the Msh proteins involved in mismatch repair. Mutation of conserved amino acids in the NTP binding and putative helix-turn-helix domains of Msh5p abolish function but are still capable of interaction with Msh4p, suggesting that NTP binding plays a role downstream of hetero-oligomer formation. No hetero-oligomers are observed between the mismatch repair MutS proteins (Msh2p and Msh6p) and either Msh4p or Msh5p. These results indicate that one level of functional specificity between the mismatch repair and meiotic crossover MutS homologs in yeast is provided by the ability to form distinct hetero-oligomers.