We have previously demonstrated that the IRA1-encoded protein inhibits the function of the RAS protein in a fashion antagonistic to the function of the CDC25 protein in the RAS-cAMP pathway in Saccharomyces cerevisiae. In an attempt to identify genes involved in the regulation of this pathway, high-copy-number plasmid suppressors of the heat shock sensitivity of the ira1 mutation were isolated. One such suppressor, MSI1, was found to encode a putative protein of 422 amino acids that shows homology to the beta subunit of the mammalian guanine nucleotide-binding regulatory proteins. Overexpression of the MSI1 gene could suppress the heat shock sensitivity and the defect in sporulation caused by the ira1 and RAS2Val19 mutations but not those of the bcy1 mutation. Furthermore, the high level of intracellular cAMP in ira1 and RAS2Val19 cells was reduced by the MSI1 gene carried on a YEp-based plasmid. These results suggest that the MSI1 protein is a negative regulator of the RAS-mediated induction of cAMP in S. cerevisiae.