Regulation of effector functions by muscarinic acetylcholine receptor subtypes is mediated by pertussis toxin-sensitive and -insensitive G proteins. In membranes from human embryonic kidney 293 cells transfected with m1, m2, and m3 muscarinic acetycholine receptors, we detected the pertussis toxin-sensitive G proteins Gi1, Gi2, and Gi3 and the pertussis toxin-insensitive G proteins Gq/11 and Gs. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled with [alpha-32P] GTP azidoanilide, in the absence and presence of carbachol, revealed the selective coupling of activated muscarinic receptors to G protein subtypes. Gq/11 was activated via m1 and m3 receptors and Gi2 was activated via m2 receptors. All three receptors subtypes mediated the activation of Gi1 and Gi3. Effective activation of Gi1 and Gi3 via m1 and m3 receptors occurred only at high carbachol concentrations (EC50 about 10-20 microM), whereas carbachol with higher potency (EC50 about 1 microM) induced activation of all G1 subtypes via m2 receptors. Thus, coupling of muscarinic receptors and G protein subtypes was principally selective; however, activation of distinct G protein subtypes by different muscarinic receptors occurred with different efficacies.