Both the α and βΓ subunits of heterotrimeric guanine nucleotide- binding proteins (G proteins) communicate signals from receptors to effectors. GβΓ subunits can regulate a diverse array of effectors, including ion channels and enzymes. Gα subunits bound to guanine diphosphate (Gα-GDP) inhibit signal transduction through GβΓ subunits, suggesting a common interface on GβΓ subunits for Gα binding and effector interaction. The molecular basis for interaction of GβΓ with effectors was characterized by mutational analysis of Gβ residues that make contact with Gα-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-β2, and β- adrenergic receptor kinase revealed the Gβ residues required for activation of each effector and provides evidence for partially overlapping domains on Gβ for regulation of these effectors. This organization of interaction regions on Gβ for different effectors and Gα explains why subunit dissociation is crucial for signal transmission through GβΓ subunits.