Abstract4,4'-Diisothiocyanato-stilbene-2,2'-dlsulfonic acid (DIDS) stimulates human platelets via cc ,,-adrenerglc receptor-mediated activation of protein kmase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 PM), measured as "P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-0-thio)diphosphate (200 fiM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 ,&ml), which ADP-ribosylates the a-subunits of G,'s and G,, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (SOpM), which uncouples G, from a,,-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 f 5% (n = 4) the pertussis toxin-catalyzed ["P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the a-subunits of G,a-1, G,a-2 and G,a-3. Thus, the a,,-adrenergic receptor activates PKC via a G-protein of the G,-family.