The beta-adrenergic receptor kinase (beta-ARK) specifically phosphorylates the activated form of various G protein-coupled receptors such as the beta 2-adrenergic receptor (beta 2-AR). Recently, G protein beta gamma subunits have been demonstrated to activate beta-ARK-mediated receptor phosphorylation. To further elucidate beta-ARK/G protein interactions, we have developed a direct binding assay. The direct binding of [35S]methionine-labeled beta-ARK to either brain Gi/Go or beta gamma subunits was rapid and saturable with similar Kd values of approximately 58 and approximately 32 nM, respectively. Both heterotrimeric G proteins and beta gamma subunits enhanced the initial rate of beta 2-AR and rhodopsin phosphorylation approximately 10-fold. Kinetic studies demonstrate that beta gamma enhances beta-ARK-mediated beta 2-AR phosphorylation both by decreasing the Km for the beta 2-AR approximately 4-fold and increasing the stoichiometry of phosphorylation from approximately 4 to approximately 11 mol/mol. An agonist- and ATP-dependent binding of beta-ARK to the reconstituted beta 2-AR was also demonstrated. In addition, beta-ARK binding was enhanced in the presence of both the activated beta 2-AR and beta gamma subunits suggesting the formation of a transient ternary complex consisting of beta-ARK, beta gamma, and beta 2-AR. Overall, these studies suggest that the specific association of beta-ARK with heterotrimeric G proteins may play an important role in promoting receptor/kinase interaction and subsequent receptor phosphorylation.