We have determined the relative abilities of several members of the G protein β and γ subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian β1 and γ3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for β-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of β and γ subunit types varies widely. Third, the reporter activity evoked by a particular combination of β and γ subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid βγ complex formed in the cell as determined by immunoprecipitation. These results suggest that different β and γ subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all γ subunit types for the β1 subunit type explains the preponderence of this subunit type in most G proteins.