Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.