To date direct evidence for the presence of a H-K-ATPase in the medulla comes from proton and potassium transport studies performed on K-restricted animals and K dependent ATP hydrolysis and Rb uptake in both normal and K-depleted animals. The present work examines K-dependent acidification in the medulla of rabbits on normal K diets. A membrane vesicle preparation was developed that was enriched for apical membranes derived from the renal medulla. Adenosine triphosphate (ATP)-dependent vesicular acidification was present and the extent of vesicular acidification was dependent on ambient K concentration. Moreover, ATP hydrolysis was dependent on ambient K concentration. K-dependent acidification was inhibited by the specific inhibitor of the gastric H-K-ATPase, SCH28080. However, significant acidification was observed in the absence of K that was not inhibited by SCH28080. The data suggest that an H-K-ATPase similar to the gastric H-K-ATPase is present in the renal medulla of rabbits on a normal K diet. The component of acidification and ATP hydrolysis that is independent of K concentration likely represents the previously characterized vacuolar H-ATPase.