Gβy-dependent interactions have profound effects on diverse signaling pathways. In many instances, Ga-GDP disrupts interactions between Gβy and its binding partners. The molecular basis of Gβy-dependent interactions with cellular proteins was studied by using alanine scanning mutagenesis of every amino acid of the Gβ subunit demonstrated to contact the Ga-GDP subunit. Each alanme-substituted mutant was analyzed for its ability to interact with the Ga-GDP, phosducin, and PLC-β2. As expected, some of the mutants were inhibitory but surprisingly, some of the alanine-substituted mutants exhibit enhanced activity in comparison with wildtype Gβy. Our results suggest a molecular logic for interaction between Gβy and its binding partners.