Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating H(+)-K(+)-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (pH(i)) was measured using the fluorescent, pH-sensitive dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the H(+)-K(+)-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical H(+)-ATPase, basolateral Na(+)-K(+)-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in pH(i) induced by inhibiting basolateral Na(+)/H(+) exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical H(+)-K(+)-ATPase.