Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.