The effect of acetazolamide (ACZ) on renal metabolism and ammoniagenesis was studied in the dog in vivo and in vitro. ACZ was administered to 10 dogs with normal acid-base status and five with chronic metabolic acidosis induced by NH4Cl. In both groups of dogs, the acute administration of ACZ markedly reduced the urinary excretion of ammonium (from 33 to 10 in normal dogs and from 100 to 23 mumoles/100 ml GFR in acidotic dogs) whereas its release into the renal vein was increased in a reciprocal fashion (from 69 to 95 in normal dogs and from 91 to 152 mumoles/100 ml GFR in acidotic dogs). ACZ did not change the total ammonium production nor the renal glutamine utilization. The renal utilization or production of glutamate, alphaketoglutarate, alanine and citrate also remained unchanged. Despite a marked urinary alkalinization, citraturia remained minimal. However, the renal cortical concentrations of glutamine, glutamate, succinate, fumarate, malate, aspartate and phosphoenolpyruvate fell following ACZ administration, especially in acidotic dogs showing rapid renal utilization of glutamine. ACZ had no effect on the same metabolites in the kidney of normal dogs even when lactate utilization was enhanced by lactate infusion. This study demonstrates that an accelerated ammoniagenic flux can proceed in the dog kidney without the renal cortical changes produced by metabolic acidosis in this species. In vitro, using dog tubules, a selective effect of ACZ on glutamine metabolism as compared to lactate was observed. ACZ reduce the rate of the reactions catalyzed by alphaketoglutarate dehydrogenase and by succinyl CoA synthetase. Other enzymes of the ammoniagenic and gluconeogenic pathways (glutaminase, GLDH, malic enzyme, PEPCK) were not changed by ACZ. The metabolic effects of ACZ observed in the intact kidney in vivo or with tubules in vitro may be in part related to the effect of ACZ on these enzymes critical for the ammoniagenic process.