1. Amiloride, applied at millimolar concentrations, results in the blockade of K+ conductance in amphibian proximal convoluted cells (PCT), fused into giant cells. 2. Amiloride results directly in a blockade of K+ conductance that is not related to inhibition of the Na(+)-H+ antiport, which would lower intracellular pH, adversely affecting K+ conductance. On the contrary, high amiloride concentrations promote entry of this lipophilic base in the cell, leading to higher cell pH. 3. Under voltage clamp conditions, control vs. amiloride, current-voltage curves from PCT fused giant cells intersect at -86.2 +/- 3.4 mV, a value close to the equilibrium potential for potassium. 4. Hexamethylene amiloride, 10(-5) M, irreversibly depolarizes the membrane potential. 5. Barium decreased by 50% the initial slope of realkalinization, following removal of a solution containing NH4Cl, as did amiloride. In addition, these blockers reduced membrane conductance by 40%, suggesting that a fraction of the amiloride-suppressible NH4+ efflux may be conductive. 6. Amiloride does not directly inhibit the Na(+)-K+, ATPase in our preparation, contrary to the prevalent belief. 7. In vivo studies show that amiloride interferes with an apical K+ conductance but it does not alter basolateral K+ conductance.