G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.