Ammonia production by rat renal cortical mitochondria was studied with both in vivo and in vitro manipulation of potassium to further elucidate the mechanisms relating potassium homeostasis and renal ammonia production. Mitochondria from potassium-depleted animals demonstrated an increase in ammonia production at all glutamine concentrations studied, which ranged from 0.5 to 10 mM. This increase in ammoniagenesis compared favorably in degree of change with and was of sufficient absolute magnitude to entirely account for the findings observed both in renal cortical slices studied in vitro and in the intact animal. When rotenone is added to the medium, increased ammoniagenesis is still detected, indicating that either glutamine entry into the mitochondria and/or the activity of phosphate-dependent glutaminase are critical rate-controlling steps. In contrast to studies with renal slices, a decrease in ammonia production was not apparent with cortical mitochondria from chronically potassium-loaded animals. In vitro alterations of the potassium homeostasis. Therefore, these experimental manipulations either do not activate an effector mechanism which takes place in vivo, or the alteration in ammoniagenesis requires an adaptation over time that is not achieved with this in vitro approach.