Pleckstrin homology (PH) domains are discrete structural modules present in numerous proteins involved in signal transduction processes. In the case of the beta-adrenergic receptor kinase (betaARK), PH domain-mediated binding of two ligands, the betagamma subunits of heterotrimeric G proteins (Gbetagamma) and phosphatidylinositol 4,5-bisphosphate (PIP2), has been shown to be required for the kinase function. In this study, the ability of Gbetagamma and PIP2 to affect membrane localization of betaARK is used to define the ligand binding characteristics of the betaARK PH domain. The binding of these ligands to the PH domain of the intact kinase is shown to be cooperative, Gbetagamma increasing the affinity of the PH domain for PIP2. Notably, although PIP2-dependent membrane association of betaARK is observed at high concentrations of this lipid, in the absence of Gbetagamma, no receptor phosphorylation is observed. Peptides derived from the receptor intracellular loop inhibit the receptor phosphorylation without affecting the membrane translocation of the kinase complex, suggesting that betaARK activity does not necessarily correlate with the amount of betaARK associated with the membrane. These results point to a distinct role for each PH domain ligand in betaARK-mediated receptor phosphorylation. Strikingly, the ligand binding characteristics of the isolated betaARK PH domain fused to glutathione S-transferase are very different from those of the PH domain of the intact kinase. Thus, in contrast to the native protein, the isolated PH domain binds Gbetagamma and PIP2 independently and with no apparent cooperativity. That protein environment plays an important role in determining the ligand binding characteristics of a particular PH domain highlights the potential risks of inferring mechanisms from studies of isolated PH domains.