A method to study (\left[\alpha^{32}\right.) P (]) GTP binding to the (\alpha) subunit of GTP-binding proteins in rat brain membranes is described. This method measures receptor-stimulated GTP binding to individual (\alpha) subunits. GTP binding is associated with two protein bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands, (40-) and (45-\mathrm{kDa}) in size, comigrate with the (\alpha) subunits of Gi/Go and Gs, respectively. Binding of (\left[\alpha-{ }^{32}\right.) P]GTP is saturable and (\mathbf{M g}^{\mathbf{2 +}})-dependent. Nucleotides compete with (\left[\alpha-{ }^{32} \mathrm{P}\right]) GTP binding in the following order: GTP (>) GDP (>) Gpp(NH)p (>) App(NH)p. Dopamine stimulates (\left[\alpha_{-}{ }^{32} \mathrm{P}\right]) GTP labeling of the 40 - and (45-\mathrm{kDa}) bands. A binding increase of (300-400 %) is observed at (10 \mu \mathrm{M}) dopamine. Isoproterenol ( (10 \mu \mathrm{M}) ) stimulates [ (\alpha) ({ }^{32}) PJGTP binding only to the (45-\mathrm{kDa}) protein band. The effects of dopamine and isoproterenol are blocked by their respective receptor antagonists, fluphenazine and propranolol. The individual (G) proteins activated by dopamine are resolved by immunoprecipitation of stimulated (\left[\alpha_{-}{ }^{32} P\right]) GTP binding to (G \alpha) s, G (\alpha) i, and G (\alpha) o with specifc anti-G (\alpha) antisera. Dopamine stimulates (\left[\alpha_{-}{ }^{32}\right.) P]GTP binding to (G \alpha) s and G (\alpha) i while the labeling of G (\alpha) wo was not significantly changed. Pertussis toxin-mediated ADP ribosylation prevents the activation of G (\alpha) i which is mediated by dopamine receptor stimulation. The methods described are useful in defining the coupling of specific neurotransmitter receptors to specific (G) proteins in native membranes. These procedures also allow measurements of receptor stimulation of individual (G) proteins in intact biological membranes. 1993 Academic Press. Inc.