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