Hydrogen ion secretion in the kidney is thought to be mediated in part by an N-ethylmaleimide (NEM)-sensitive proton-translocating adenosine triphosphatase (ATPase). This enzyme has been found throughout the nephron, but it has not been completely characterized enzymatically in the rat collecting duct. In the present study we characterized the NEM-sensitive ATPase from microdissected cortical (CCT) and medullary (MCT) collecting tubules of the rat nephron. At optimum conditions, NEM-sensitive ATPase activity was the same in both tubule segments: activity was 275.6 +/- 18.6 pmol/mm/h in the CCT and 280.3 +/- 35.2 pmol/mm/h in the MCT (n = 23, NS). ATP sensitivity was greater in CCT than in MCT, and in the former guanosine triphosphate was able to partially support enzyme activity. Maximal enzyme inhibition with NEM occurred at a lower concentration in CCT as compared to MCT. At pH 7.0 in MCT enzyme activity was approximately one half that seen at pH 7.4; in MCT and CCT, the pH optimum was 7.4. The temperature optimum in both segments was between 37 and 42 degrees C. Enzyme activity in CCT and MCT was linear to 30 min and proportional to tubule length. These results demonstrate that there are important differences in the NEM-sensitive ATPase isolated from two segments of rat collecting duct, and raise the possibility that enzyme heterogeneity may exist.