Hippurate is known to be synthesized from benzoate and glycine in the liver and kidney. It takes part in renal ammoniagenesis by modulating the activity of gamma-glutamyl transpeptidase (gamma GT). Due to its chemical structure, however, hippurate might also serve as a substrate of renal ammoniagenesis. Hippurate may yield ammonia either having been cleaved by hippuricase or by Erlenmeyer's reaction after condensation with an aldehyde. In order to elucidate the possibility of hippurate being a substrate of renal ammoniagenesis, experiments were carried out on cortical kidney slices and on isolated tubular segments of the rat. The incubation medium (pH 7.1) was enriched with 10 mmol/l hippurate spiked with 15N-hippurate, some of the known competitive inhibitors of hippuricase, acivicin and different aldehydes. Factors known to affect hippuricase or gamma GT did not interfere with renal ammonia production. Glyceraldehyde (up to 1.0 mmol/l) but not glycerate had a stimulating effect, especially on the ammoniagenesis from hippurate. In normal rats fed a vegetarian diet, 1% of the added 15N moiety was found to be 15NH3. Renal 15NH3 production was significantly greater if, prior to the experiments, the animals were either acidotic or had a reduced renal mass or were fed animal proteins. These results indicate that hippurate may, to a certain extent, serve as substrate for ammoniagenesis.