In humans and animals, the administration of Li or amiloride results in a defect in urinary acidification. Both agents are thought to cause this by a voltage-dependent mechanism in the distal nephron. This study was designed to determine the effects of chronic Li and amiloride administration on the two main transport enzymes in rat nephron collecting tubule, the Na-K-adenosine triphosphatase (ATPase) and the H(+)-ATPase. We also examined the effects of both agents on these enzymes in vitro. Amiloride administration resulted in a decrease in Na-K-ATPase and H(+)-ATPase activities in cortical collecting tubule and medullary collecting tubule. Therapeutic concentrations of amiloride in vitro inhibited Na-K-ATPase activity, but only in cortical collecting tubule. The effects of Li administration were different; it decreased Na-K-ATPase and H(+)-ATPase in both cortical collecting tubule and medullary collecting tubule. In cortical collecting tubule, the inhibitory effect on H(+)-ATPase activity was seen in vitro at a Li concentration similar to that found in urine. In contrast to the effect of Li on the H(+)-ATPase, in vitro Li stimulated Na-K-ATPase activity. These results suggest that the mechanism of action whereby these two agents result in distal renal tubular acidosis in humans and animals are different. In the collecting tubule, amiloride appears to act solely through a voltage-dependent mechanism by inhibiting cortical collecting tubule Na-K-ATPase. Li, by contrast, appears to have an additional effect in the cortical collecting tubule to inhibit the H(+)-ATPase. The biochemical differences seen with these drugs may explain the more severe acidemia universally found in animals after chronic Li administration.