Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of α from βγ subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of Gβγ. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked Gβγ stimulation of adenylyl cyclase 2, phospholipase C-β3, potassium channels, and β-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between Gβγ and Gαo. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and β-adrenergic receptor kinases that participate in Gβγ interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from Gβγ.