Ammonia production and secretion by the proximal tubule accounts for most of the ammonia that appears in the urine. Rates of ammonia production and net luminal ammonia secretion were measured in isolated perfused mouse proximal tubule segments. This approach combines the in vitro microperfusion technique with a sensitive bioluminescence assay for total ammonia and permits the determination of ammonia production and secretion rates in specific proximal tubule segments bathed and perfused with defined solutions. Luminal perfusion stimulates ammonia production by proximal tubule segments in a flow-related manner. The effect of perfusion is not dependent on intact Na⁺-H⁺ exchange. In contrast, the rate of net luminal secretion of ammonia is largely dependent on Na⁺-H⁺ exchange but not markedly dependent on an acid luminal fluid pH. These results suggest an important role of Na⁺-NH₄⁺ exchange in the mechanism by which the Na⁺-H⁺ exchanger facilitates net ammonia secretion.