The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of the beta 2-adrenergic receptor (beta 2AR) and related G protein-coupled receptors. To further elucidate the role of beta ARK in receptor desensitization, we generated a beta ARK dominant negative mutant by converting an invariant lysine residue in the protein kinase catalytic domain to an arginine. Expressed and purified beta ARK-K220R was able to inhibit wild type beta ARK phosphorylation of the beta 2AR in vitro. When stably transfected into human bronchial epithelial BEAS-2B cells, beta ARK-K220R promoted a > 2-fold increase in beta-agonist-stimulated cAMP production without affecting beta 2AR sequestration. In contrast, beta ARK-K220R had no effect on the desensitization of the prostaglandin E2 receptor response in BEAS-2B cells. These findings directly demonstrate a role for beta ARK in desensitization of the beta 2AR in intact cells and establish the potential utility of using dominant negative mutants to elucidate the substrate specificity of G protein-coupled receptor kinases.