G protein-coupled receptor kinases (GRKs) such as rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) play an important role in agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK5 is a recently identified member of the GRK family that has greater homology with rhodopsin kinase than with beta ARK. To further characterize the activity of GRK5, it has been overexpressed in Sf9 insect cells and purified by successive chromatography on S-Sepharose and Mono S columns. GRK5 phosphorylates the beta 2-adrenergic receptor (beta 2AR), m2 muscarinic cholinergic receptor, and rhodopsin in an agonist-dependent manner to maximal stoichiometries of approximately 2.5, 1.5, and 1 mol of phosphate/mol of receptor, respectively, with Km values of approximately 0.5 microM for the beta 2AR, approximately 16 microM for rhodopsin, and approximately 24 microM for ATP. Peptide phosphorylation studies suggest that in contrast to beta ARK and rhodopsin kinase, GRK5 preferentially phosphorylates on nonacidic peptides with a Km of approximately 1.5 mM. Heparin and dextran sulfate were found to be potent inhibitors of GRK5 with IC50 values of approximately 1 nM, thereby being at least 150-fold more potent on GRK5 than on beta ARK. GRK5 can also be activated by polycations, with 10 microM polylysine promoting an approximately 2.6-fold activation. Overall, these studies demonstrate that GRK5 has unique properties that distinguish it from other members of the GRK family and that likely play an important role in modulating its mechanism of action.