Abstract Hormone receptor complexes are tight complexes and rather long-lived. Thus several cycles of activation and deactivation of adenylate cyclase might conceivably occur while the hormone is bound. Moreover, β-adrenergic receptors can become desensitized. Regulatory steps are therefore postulated which control adenylate cyclase activity and in addition might also modulate the hormone receptor interaction. Isoprenaline ( d,l-isoproterenol) stimulated adenylate cyclase from pigeon erythrocyte membranes is synergistically activated by analogs of GTP: Guanylylimidodiphosphate (Gpp(NH)p), Guanylylmethylenediphosphonate (Gpp(CH 2)p) and Guanosine-5′-O-(3-thiotriphosphate) (GTPγS). Among several photoaffinity labels of GTP synthesized, GTP-γ-azidoanilide ( ) was best suited for the identification of GTP-binding proteins in pigeon erythrocyte membranes, because it binds with high affinity (K d ∼ 3 × 10 −7 M) and activates adenylate cyclase about one-half as effectively as Gpp(NH)p. Four major protein fractions were labeled in membranes on photoactivation of GTP-γ-azidoanilide. In solubilized membranes which contain adenylate cyclase fully responsive to guanylnucleotide activation, only two guanylnucleotide binding proteins were reactive with the photoaffinity derivative of GTP. These proteins had molecular weights of 42,000 and 23,000, respectively. Soluble guanylnucleotide binding proteins were removed by affinity chromatography with Sepharose-NH-(CH 2) 3-CO-NH- -NH-pppG with loss of guanylnucleotide and fluoride activation. Readdition of the protein fraction released from the affinity column restored guanylnucleotide and fluoride activation. This suggests the existence of separable protein moieties which are required for adenylate cyclase activation.