The pleckstrin homology (PH) domain is an approximately 100-amino-acid region of sequence homology present in numerous proteins of diverse functions, which forms a discrete structural module. Several ligands capable of binding to PH domain-containing proteins have been identified including phosphatidylinositol 4,5-bisphosphate (PIP2) and the G beta gamma subunits of heterotrimeric G proteins (G beta gamma), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of G beta gamma and lipid to the PH domain of the beta-adrenergic receptor kinase (beta ARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase. PIP2 and to a lesser extent phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidic acid were the only lipids tested capable, in the presence of G beta gamma, of enhancing beta ARK activity. In contrast, the Km and Vmax for phosphorylation of a soluble beta ARK substrate (casein) was not altered in either the presence or absence of G beta gamma and/or PIP2. A fusion protein of the beta ARK containing an intact PH domain inhibits G beta gamma/PIP2-dependent beta ARK activity. In contrast, a mutant fusion protein in which a tryptophan residue, invariant in all PH domain sequences, is mutated to alanine shows no inhibitory activity. The requirement for the simultaneous presence of two PH domain binding ligands represents a previously unappreciated mechanism for effecting membrane localization of a protein and may have relevance to other PH domain-containing proteins.