Using the rat cerebral cortical membranes that had once been frozen and stored at -80 degrees C, the stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding mediated through alpha(2)-adrenergic receptors (alpha(2)-ARs) was pharmacologically characterized. The stimulatory effects of (-)-noepinephrine ((-)-NE) and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14,304) at maximally effective concentrations were prominent only in the presence of micromolar to submillimolar levels of GDP, with the greatest signal/noise ratio achieved at 100 and 30-100 microM GDP for (-)-NE and UK-14,304, respectively. The alpha(2)-AR-mediated [(35)S]GTPgammaS binding was also critically dependent on the presence of millimolar concentrations of MgCl(2) in assay medium. The maximum stimulation induced by UK-14,304 was equivalent to, or even greater than, that of an endogenous ligand (-)-NE at lower concentrations of NaCl, while it became a partial agonist with higher concentrations of NaCl. Concentration-response curves for several agonists revealed that the omission of NaCl from incubation buffer generally shifted the curves leftward and increased the relative efficacies as compared to the endogenous ligand. The pharmacological profile characterized with a series of adrenergic agonists and antagonists indicated that this response was derived from activation of G proteins coupled with alpha(2)-ARs, in particular alpha(2D)-AR subtype, though the possible involvement of alpha(2C)-ARs was not completely excluded. However, the stimulatory effects of several adrenergic compounds, such as rauwolscine and yohimbine, were ascribed to their agonist properties at 5-HT(1A) receptors. This method serves as a simple but useful strategy for investigating the functional interaction between alpha(2D)-ARs and their coupled G proteins in rat native cerebral cortical membranes, especially in the field of psychopharmacology and biological psychiatry.