In rat terminal inner medullary collecting duct (tIMCD), the Na,K-ATPase mediates NH₄⁺ uptake, which increases secretion of net H⁺ equivalents. K⁺ and NH₄⁺ compete for a common binding site on the Na,K-ATPase. Therefore, NH₄⁺ uptake by the Na,K-ATPase should be augmented in hypokalemia because interstitial K⁺ concentration is reduced. We asked if the Na,K-ATPase is also upregulated during hypokalemia and if increased Na⁺ pump expression leads to increased NH₄⁺ uptake across the basolateral membrane. To induce hypokalemia, rats ate a diet with low or normal K⁺ content for 3 or 7 days. In IMCD cells from rats given 3 days of dietary K⁺ restriction, ß1 subunit protein expression was increased, although changes in 1 subunit protein expression and Na,K-ATPase activity were not detected. However, after 7 days of the K⁺-restricted diet, an increase in both 1 and ß1 protein expression as well as Na,K-ATPase activity were observed. The magnitude of Na,K-ATPase-mediated NH₄⁺ uptake across the basolateral membrane (JNH₄⁺) was determined in tIMCD tubules perfused in vitro from rats eating either the normal or K⁺-restricted diet for 3 days. JNH₄⁺ was the same in tubules from rats on diets with either low or normal K⁺ content when perfused at the same extracellular K⁺ concentration. However, JNH₄⁺+ was reduced > 60% when K⁺ concentration was increased from 10 to 30 mM in tubules from rats in either treatment group. In conclusion, while hypokalemia upregulates expression of the 1 and ß1 subunits of the Na,K-ATPase and Na,K-ATPase activity in rat IMCD, with 3 days of K⁺ restriction, NH₄⁺ uptake by the Na,K-ATPase is increased to a greater extent because of the reduced K⁺ concentration observed in the interstitium of the inner medulla in vivo.