The role of Na+ and H+ in the regulation of D2 receptor affinity for ligands was studied to determine the molecular mechanisms of this phenomenon. The potency of substituted benzamide derivatives and agonists at D2 receptors depended on the concentration of Na+ and H+, whereas the potency of other antagonists was relatively unaltered by changes in pH or Na+ concentration. The potency of agonists was generally decreased in the presence of NaCl or lowered pH. For example, in the absence of sodium the affinity of D2 receptors for dopamine was decreased 17-fold by lowering of the pH from 8.0 to pH 6.8. Addition of NaCl caused 2-4-fold decreases in affinity for most agonists. The affinity of the receptors for two substituted benzamide derivatives, on the other hand, was reduced 6-44-fold by elevated concentrations of H+ but was enhanced 7-24-fold in the presence of Na+. The regulation by H+ of the potency of dopamine was selective for D2 receptors, because binding of dopamine to neostriatal D1 receptors was unaffected by changes in pH. Decreasing of the pH from 8.0 or 7.3 to 6.8 facilitated the dissociation of the substituted benzamide ligand [125I]epidepride from D2 receptors but inhibited dissociation of [3H]spiperone. Furthermore, the presence of NaCl or lowered pH slowed inactivation of D2 receptors by N-ethylmaleimide. Together, these data suggest that the conformation of D2 receptors is regulated by both Na+ and H+. The affinity of D2 receptors for agonists and substituted benzamide antagonists varies according to the conformational state of the receptors, whereas other antagonists bind to both forms with approximately equal potency. Amiloride is a compound that interacts with many sodium-binding macromolecules. At equilibrium, amiloride inhibited the binding of [3H]spiperone and [125I]epidepride in a manner suggesting a more complex interaction than simple competitive inhibition. The rate of dissociation of both radioligands was enhanced by amiloride, as would be expected for allosteric inhibition of binding. The sensitivity of D2 receptors to pH, sodium, and amiloride may be a reflection of the ability of D2 receptors to modulate Na+/H+ exchange.