The β-adrenergic receptor kinase (β-ARK), which specifically phosphorylates only the agonist-occupied form of the β-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The β-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the β 2 -adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that β-ARK may be the first sequenced member of a multigene family of receptor kinases.