To evaluate the contribution of the purine nucleotide cycle (PNC) in renal ammoniagenesis, ammonia production (AP) in cortical tubular suspensions and microdissected nephron segments of control and acidotic rats was determined using various amino acids, including glutamine (Gln) and aspartate (Asp). In the cortical tubular suspensions, the best substrate for ammoniagenesis was Gln (153.1 +/- 19.4 nmol.mg protein-1.15 min-1) followed by Asp (70.9 +/- 11.4). Metabolic acidosis resulted in a significant increase of AP only from Gln (316.5 +/- 36.1 nmol.mg protein-1.15 min-1, P less than 0.01 vs. control). Intranephron distribution of AP (pmol/mm or a glomerulus/15 min) from Gln showed that the first segment of the proximal tubule (S1) was highest in control (95.7 +/- 9.0), and its AP markedly increased in acidosis (221.6 +/- 8.3, P less than 0.001 vs. control). The most interesting and striking finding was that with Asp as a substrate, AP was maximal in S1 (165.0 +/- 32.8), with a value exceeding that from Gln. An adenylosuccinase inhibitor, 6-mercaptopurine (0.1 mM), significantly inhibited AP from Asp in S1 and S3, and from Gln in S1. On the contrary, a specific inhibitor of phosphoenolpyruvate carboxykinase, 3-mercaptopicolinate (0.1 mM), caused a significant decrease of AP from Gln, but not from Asp, in S1. From these results it could be concluded that AP via PNC can occur at high rates, especially in S1, only when Asp is present at high concentrations.