The properties of alpha 2-adrenergic receptor binding sites and the receptors inhibiting [3H]norepinephrine (3H-NE) release from slices of cerebral cortex were compared. [3H]RX 781094, an alpha 2-adrenergic receptor antagonist radioligand, labeled a single class of binding sites in membranes at 37 degrees with the pharmacological specificity expected of alpha 2-adrenergic receptors. 5'-Guanylimidodiphosphate (Gpp(NH)p) and NaCl caused small increases in the potencies of antagonists at the 3H-RX binding sites but decreased the potencies of agonists 4-22-fold. Antagonists blocked the inhibition of potassium-evoked 3H-NE release caused by exogenous NE with potencies similar to those in competing for 3H-RX binding sites. Partial receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine whether there was a receptor reserve. EEDQ dose-dependently decreased both the density of 3H-RX binding sites and the maximal inhibition of 3H-NE release by different agonists. For most agonists, KA values calculated after the receptor inactivation did not differ significantly from EC50 values; however, for epinephrine a small receptor reserve was apparent. The proportion of 3H-RX binding sites lost was similar to the proportion of functional receptors lost after EEDQ treatment. The functional KA values for agonists correlated most closely with KD values for the low affinity binding state observed in the presence of Gpp(NH)p and NaCl. However, both epinephrine and NE still showed two-site binding curves under these conditions, and it was the high affinity subpopulation of sites observed in the presence of Gpp(NH)p and NaCl which resembled most closely the functional KA values. These data suggest that 3H-RX labels binding sites with properties similar to the alpha 2-adrenergic receptors inhibiting 3H-NE release in cerebral cortex. There is little or no receptor reserve for this effect, and there appears to be a binding state for the natural catecholamine agonists which has an affinity lower than that for mediating this functional response.