The effect of the sulfhydryl alkylating agent N-ethylmaleimide on the modulation of potassium-evoked [(3)H]noradrenaline release via inhibitory presynaptic receptors was studied using synaptosomes from rabbit hippocampus. Dose-response curves for the ?(2)-adrenoceptor agonist clonidine, the preferential ?-opioid receptor agonist ethylketocyclazocine and the A(1)-adenosine receptor agonist (?)phenylisopropyladenosine were compared to the effects of these agonists after pretreatment of [(3)H]noradrenaline loaded synaptosomes with N-ethylmaleimide (2 ?M) for 15 min. The inhibitory effects of all three agonists were attenuated to the same extent in a non-competitive manner after pretreatment with N-ethylmaleimide. Pertussis toxin-catalyzed [(32)P]ADP-ribosylation of synaptosomal proteins after purification of synaptosomes on a discontinuous Percoll gradient revealed the presence of three toxin-sensitive G proteins with apparent molecular weights of the ?-subunits between 41 and 39 kDa. N-Ethylmaleimide treatment of synaptosomes prior to pertussis toxin-catalyzed [(32)P]ADP-ribosylation reduced the incorporation of radioactivity into the toxin substrates to an extent comparable to the invalidation of agonist-induced inhibition of [(3)H]noradrenaline release. The quantitative agreement of the effects of N-ethylmaleimide on the modulation of [(3)H]noradrenaline release and on pertussis toxin-catalyzed [(32)P]ADP-ribosylation lends support to the proposal that inhibitory receptors on noradrenergic terminals in rabbit hippocampus are coupled to pertussis toxin-sensitive G proteins. The observation that the extent of functional antagonism after N-ethylmaleimide pretreatment was the same for all three agonists investigated is compatible with the existence of a common step in the signal transduction mechanism of the three pharmacologically different receptors, presumably on the level of a common G protein.