Aldosterone stimulates not only Na+ absorption but also urinary acidification. In this investigation the effects of aldosterone on H+ transport are examined in vitro in turtle bladder, a urinary membrane in which several of the factors controlling H+ transport have been defined. H+ transport was increased in bladder halves exposed to aldosterone compared to control halves. Stimulation of H+ secretion was observed as early as 1 h after addition of aldosterone and occurred before that of Na+ transport. In bladders depleted of endogenous substrate addition of glucose increased H+ transport more in aldosterone-treated halves (10.0+/-1.3 nmol/min) than in control halves (6.8+/-2.3). Addition of pyruvate failed to increase H+ transport (--0.3+/-0.7) in control halves but caused significant increments (2.4+/-0.5) in aldosterone-treated halves. In aldosterone-treated bladders glucose caused larger increments (16.5+/-2.7) in H+ transport than pyruvate (9.3+/-2.0) when halves of the same bladders were compared. Na+ transport, however, was equally increased by the two substrates. Despite the differences in time course and substrate requirements between the stimulation of H+ and Na+ transport, both increases were abolished by actinomycin-D. To examine the effect of aldosterone on the force of the H+ pump, protonmotive force, the pH gradient that would nullify the transport rate was determined with and without aldosterone. Aldosterone did not alter protonmotive force but significantly increased the slope of the H+ transport rate on the applied pH gradient. It is concluded that aldosterone stimulates H+ transport independently of Na+ transport. It increases the responsiveness of the transport rate to glucose and to a lesser extent pyruvate, an effect probably secondary to the increased transport rate. Equivalent circuit analysis indicates that aldosterone facilitates the flow of protons through the active transport pathway but does not increase the force of the pump.