The [35S]GTPgammaS binding assay represents a functional approach to assess the coupling between receptors and G-proteins. The optimal conditions for [35S]GTPgammaS binding to human brain homogenates were established in postmortem samples of prefrontal cortex. The influence of protein content, incubation time, GDP, Mg(2+), and NaCl concentrations on the [35S]GTPgammaS binding were assessed in the absence and presence of the alpha(2)-adrenoceptor agonist UK14304 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine). In conditions of 50 microM GDP and 100 mM NaCl, UK14304 increased the apparent affinity of the specific [35S]GTPgammaS binding without changing the apparent density. Concentration-response curves to agonists of alpha(2)-adrenoceptors, mu-opioid, 5-HT(1A), cholinergic muscarinic, and GABA(B) receptors displayed, in the presence of NaCl, maximal stimulations between 24% and 61% with EC(50) values in the micromolar range. Selective antagonists shifted to the right the agonist-induced stimulation curves. The G(i)/G(o)-protein alkylating agent N-ethylmaleimide decreased basal [35S]GTPgammaS binding in a concentration-dependent manner and inhibited the stimulation induced by the different agonists. In cortical sections, [35S]GTPgammaS binding to gray matter was stimulated by the agonist UK14304. The present study demonstrates that functional studies of the receptor coupling to G(i)/G(o)-proteins can be performed in postmortem human brain samples.