In vivo, over a short 2-hour period, renal ammonia production in dogs increased (+41%) and renal glutamate concentrations decreased (-21%) following acid challenge. Since glutamate is an inhibitor of glutamine deamidating enzyme, glutaminase I, these results are compatible with the concept that during acid challenge, lowered concentrations of glutamate increase ammoniagenesis via accelerated glutamine deamidation. However, glutamate infusions in vivo into dogs do not alter ammoniagenesis from glutamine. To explain this paradox, evidence is presented that exogenous glutamate does not reach ammonia producing sites. In vivo, exogenous glutamate neither increases nor decreases renal ammonia production; whereas exogenous alanine, which must form glutamate through transamination in order to be deaminated, produces a good deal of ammonia. That both the amino nitrogen of glutamine and the amino nitrogen of alanine produced more ammonia than the amino nitrogen of glutamate at equimolar concentrations in isolated dog tubules suggests that there is a relative impermeability of glutamate to ammonia producing sites even in vitro. Despite this relative barrier, dog tubules do produce ammonia from glutamate allowing one to conclude that this substrate does reach ammonia producing sites in vitro. Here, exogenous glutamate added to incubation medium did depress utilization of glutamine significantly. We conclude that there may be an important role for glutamate concentrations at ammonia producing sites in regulating renal ammoniagenesis.