We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the α(2A)-adrenergic receptor (α(2A)AR). In whole-cell phosphorylation studies, recombinantly expressed human α(2A)AR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 ± 40% in COS-7 cells and 201 ± 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of α(2A)AR function, amounting to a 68 ± 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein- coupled receptor kinase activity, because PMA-promoted desensitization of a mutated α(2A)AR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type α(2A)AR. To test whether PKC phosphorylation is a mechanism by which α(2A)AR can be regulated by other receptors, the α(lb)AR was co-expressed with the α(2A)AR in Chinese hamster ovary cells. Upon selective activation of α(1b)AR, the function of α(2A)AR underwent a 53 ± 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the α(2A)AR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the α(2A)AR and other receptors can occur.