Most G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation followed by arrestin binding and internalization. In this study we explored the time-, ligand-, and concentration dependence of alpha(2)-adrenoceptor internalization in human embryonal kidney (HEK-293) cells expressing alpha(2A)- and alpha(2B)-adrenoceptors. We also explored the relationship between ligand-induced receptor internalization and agonist efficacy, determined with a [S-35]GTPgammaS binding assay. The results showed rapid dose-dependent internalization of both alpha(2A)- and alpha(2B)-receptors; the extent of internalization was directly proportional to agonist efficacy. The agonist UK 14,304 had a subtype-specific high efficacy at alpha(2A)-AR and dexmedetomidine at alpha(2B)-AR. Agonist-induced [S-35]GTPgammaS binding was totally blocked by pretreatment with pertussis toxin (PTX) for both receptor subtypes, while only about 50% of the internalization was blocked by PTX. The results indicate that the extent of internalization of alpha(2A)-AR and alpha(2B)-AR is proportional to agonist efficacy, but only partly dependent on G(i) protein coupling. (C) 2004 Elsevier Inc. All rights reserved.