We studied changes in renal mitochondrial glutamine metabolism during the development of and recovery from K+ depletion in rats. Significant increase in mitochondrial NH3 production was noted after 3 days of K+-free diet. Ammoniagenesis in K+-depleted animals reached maximal level within 2 wk of K+ deprivation when there was 64% increase in NH3 production. In contrast to the pattern of changes in mitochondrial ammoniagenesis, phosphate-dependent glutaminase (PDG) activity increased within the first 48 h of K+ deprivation, before there was any increase in NH3 production, and did not plateau even after 2 wk of K+-free diet. The disparity between NH3 production and PDG activity cannot be explained by the difference in matrix glutamate level, thus raising the possibility that mitochondrial glutamine entry may be a rate-limiting step for ammoniagenesis during K+ depletion. Recovery from K+ depletion was studied in animals prefed with K+-free diet for 2 wk prior to the initiation of K+-supplemented diet. Muscle K+ content of K+-depleted animals returned to normal after 1 wk of K+ replacement. Mitochondrial NH3 production decreased concomitantly with the attenuation in K+ deficit but did not reach the base-line value even after K+ deficit was completely corrected. Additional experiments with isolated cortical tubules also showed persistent increase in NH3 production after the correction of K+ deficit. Thus, unlike earlier studies in rats during the recovery from metabolic acidosis, which showed only increased ammoniagenesis in isolated mitochondria but not in cortical slices, animals recovered from K+ depletion demonstrated augmented NH3 production both in isolated mitochondria and intact renal tissues.