We measured agonist-stimulated binding of the stable GTP-analog guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) as a functional assay to monitor G-protein activation by recombinant human alpha2-adrenoceptor subtypes alpha2A, alpha2B and alpha2C. (-)-Noradrenaline was a full agonist in all receptors. Dexmedetomidine, UK14,304, clonidine and oxymetazoline showed subtype-selectivity in efficacy. Dexmedetomidine was a full agonist at alpha2B and a partial agonist at alpha2A; UK14,304 was a full agonist at alpha2A and a partial agonist at alpha2B. Clonidine and oxymetazoline were weak partial agonists at the alpha2B-subtype, but appeared inactive at alpha2A and alpha2C. The [35S]GTPgammaS binding assay provides functional information on pertussis toxin-sensitive G-protein activation, complementing radioligand binding assays and conventional second messenger assays.