The contribution of amino acids other than glutamine to renal ammoniagenesis was investigated in renal tubules obtained from control rats and rats with metabolic acidosis by incubating 2.5 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, [15N]aspartate, [15N]alanine, or [15N]glycine, either as the sole N source in Krebs bicarbonate buffer or as a labeled substrate in a medium containing a mixture of unlabeled amino acids. With control tissue in Krebs buffer, approximately 75% of total ammonia was derived from 5-N of glutamine, whereas 2-N of glutamine, glutamate, aspartate, alanine, and glycine supplied 1, 10, 13, 4, and 18%, respectively. In the acidotic state, these values were 51, 30, 30, 30, 10, and 15% of the total ammonia produced, respectively. The ammonia that could not be accounted for by 15N analysis was derived from endogenous sources. Studies with tubules incubated in Krebs medium alone indicated that, in both control and acidosis, the calculated fraction of ammonia derived from endogenous sources was significantly decreased by addition of either 0.7 or 2.5 mM glutamine. However, ammonia production from endogenous sources was similar whether 0.7 or 2.5 mM glutamine was used as a sole exogenous substrate. Incubations of control tissue in buffer supplemented with an amino acid mixture revealed a significant decrease in ammonia production from [5-15N]glutamine compared with incubation in Krebs buffer alone. In chronic acidosis, no significant difference was found in total ammonia formation from [5-15N]glutamine compared with Krebs buffer alone.(ABSTRACT TRUNCATED AT 250 WORDS)