Saccharomyces cerevisiae expresses two RAS gene products (RAS1 and RAS2) highly homologous to mammalian p21ras which mediate glucose-stimulated cyclic-AMP formation. Mating pheromone inhibits RAS-linked adenylyl cyclase activation and this is dependent upon the alpha-factor receptor (STE2) and its associated G-protein beta-subunit (STE4). We now show that this pheromone effect is independent of mating pathway signalling components "downstream" of STE4 but displays an absolute requirement for an additional G-protein alpha-subunit encoded by GPA2. alpha-mating factor effects also involve a specific suppression of normal RAS2 activity as the constitutively activated mutant RAS2vall9 as well as wild type. RAS1 are insensitive to inhibition. Interaction between GPA2, STE4-STE18, RAS2 and adenylyl cyclase in yeast could give important insight into signalling pathways controlling normal and oncogenic p21ras activity in man.