Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric proteins that transduce extracellular signals into intracellular changes. Functionally different G proteins have been identified by their different alpha subunits. The beta and gamma subunits have been assumed to constitute a common pool shared among various G protein heterotrimers. Two gamma subunits (gamma 3 and gamma 4) have been identified through molecular cloning; these are in addition to two subunits (gamma 1 and gamma 2) that were previously characterized. G proteins purified from a variety of mammalian tissues were examined with antisera specific to three of the gamma subunits. The antisera react with different gamma subunits associated with some of the purified G proteins but not all. This demonstrates that different G protein heterotrimers from different tissues carry structurally distinct members of the gamma-subunit family. Diversity in the structure of the gamma as well as the alpha and beta subunits and preferential associations between members of subunit families increase structural and possibly functional diversity of G proteins.