To investigate the mechanism by which amiloride inhibits urinary acidification, its effects on H+ secretion were examined in the isolated urinary bladder of the fresh water turtle. In short-circuited turtle bladders amiloride inhibited H+ secretion by 30% and Na+ transport by 100%. Maximal inhibition was reached at 10(-4) M amiloride for both transport systems. In contrast to amiloride, ouabain did not affect H+ secretion despite complete inhibition of Na+ transport. In bladders first treated with ouabain amiloride failed to inhibit H+ secretion and in bladders first treated with amiloride, the inhibition of H+ secretion was partially reversed by ouabain. The inhibition of H+ secretion by amiloride is attributed to hyperpolarization of the luminal cell membrane and the imposition of a voltage opposing the movement of protons in the active transport pathway.