The influence of potassium homeostasis on ammonia production was investigated with both cortical and medullary slices from rat kidney. Renal cortical slices from rats depleted of potassium by dietary restriction produced 31% more NH3 than slices from pair-fed controls. A high-potassium diet for 1 wk diminished ammonia production in cortical slices by 5% in comparison with rats pair fed a normal diet (161 vs. 169 mumol/90 min per g wet wt; P less than 0.05). Pair feeding did not introduce an experimental artifact, since animals ingesting similar K+ diets showed no difference in NH3 production. In contrast to cortex, NH3 production by outer medullary slices from K+-depleted animals was similar to pair-fed controls. Medulla from potassium-loaded rats exhibited an impressive inhibition in NH3 production averaging 36%. These striking differences between cortex and medulla suggest that specific alterations in potassium homeostasis may influence NH3 production selectively at different tubular sites. In vitro manipulation of K+ homeostasis produced by varying bathing media K+ from 0 to 144 mM, with concomitant changes in intracellular K+ from 30 to 130 mM, had no detectable influence on NH3 production by cortical slices. Hence altered cortical ammoniagenesis is not the direct result of acute changes in extracellular or intracellular cortical fluid K+ or in the transcellular gradient for K+. Although the specific cellular mechanisms whereby K+ alters ammoniagenesis remains undefined, the observation that K+ loading diminishes while K+ depletion enhances NH3 production supports the supposition that K+ and NH3 are linked in a physiologic control system.