beta-Adrenergic receptor kinase (betaARK) plays a pivotal role in phosphorylating and desensitizing G protein coupled receptors by virtue of pleckstrin homology (PH) domain-mediated membrane translocation. betaARK is localized to the specific membrane compartment by betagamma subunits of G proteins (Gbetagamma) and phosphatidylinositol phosphates that specifically and coordinately bind to the carboxyl and amino terminus half, respectively, of the betaARK PH domain. To determine the function of the betaARK PH domain in intact cells, various point mutations were incorporated in the betaARK PH domain and the constructs were tested for their ability to agonist-dependently phosphorylate the muscarinic acetylcholine receptor or alpha-adrenergic receptor in COS-7 cells. It was found that selected mutations (i.e., W643A, L647A, and an Ala-insertion following Trp643) completely abolished betaARK's ability to phosphorylate the receptors in whole-cell labeling experiments. These residues are located in the carboxyl-terminal alpha-helix of the PH domain that is essential for binding to Gbetagamma. This site-directed mutation study provides molecular information on the mechanism and significance of the betaARK PH domain function in the intact cell system.