Treatment of smooth-muscle cells with R-phenylisopropyladenosine ( R-PIA) leads to a loss of A 1 adenosine receptor (A 1AR)-mediated inhibition of adenylate cyclase, a decrease in receptor number and an increase in receptor phosphorylation. In this study, the role of the β-adrenergic receptor kinase (βARK) in the phosphorylation and inactivation of the A 1AR was examined. A 1ARs were purified from bovine brain and reconstituted into phospholipid vesicles, with or without a 10-fold excess of G i/G o (a 50:50 mixture). The reconstituted receptor preparations were phosphorylated with βARK in the absence (control) or presence (treated) of R-PIA. R-PIA stimulated A 1AR phosphorylation by 2–3-fold over control. Phosphorylation of the A 1AR was blocked by XAC, an A 1AR antagonist, underscoring its agonist dependence. The stoichiometry of phosphorylation obtained was approx. 1.3 mol of phosphate per mol of A 1AR. Phosphorylation of the A 1AR by βARK was enhanced by an additional 42% when G βγ (30 nM) was included in the phosphorylation mixture. In order to test the role of phosphorylation on receptor function, the purified A 1AR was reconstituted with a mixture of G i/G o, phosphorylated with βARK and used to determine high-affinity [ 125I]APNEA (A 1AR agonist) binding. Agonist binding was reduced by about 50% in the treated preparations compared to control. In contrast, antagonist ([ 3H]XAC) binding was increased by about 50%. These data are consistent with an uncoupling of the A 1AR from G proteins following receptor phosphorylation. In control preparations, R-PIA stimulated GTPase activity from 0.08 to 0.164 pmol P i released/pmol G i/G o per min. Phosphorylation of receptor by βARK reduced R-PIA-stimulated GTPase activity by 35%. In addition, phosphorylation of the A 1AR by βARK decreased R-PIA-stimulated GTPγS binding by 62%. These data provide evidence that A 1AR phosphorylation by βARK results in a diminished receptor-G-protein interaction.