G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.