Plasma and muscle amino acid (AA) and ammonia (NH3) responses were measured during prolonged submaximal exercise in humans. Increased NH3 production during submaximal exercise has been attributed to the activity of the purine nucleotide cycle, without consideration of any possible contribution from AA. Six men cycled at 75% of maximal O2 uptake until exhaustion on two occasions after 2.5 days of ingestion of a high-carbohydrate or mixed diet. Plasma samples (antecubital vein) and muscle biopsies (vastus lateralis) were obtained at rest and during exercise and analyzed for plasma and muscle NH3 and AA as well as muscle metabolites. There were no significant diet effects in these parameters, so the majority of results focus on the effects of exercise. Plasma and muscle NH3 increased significantly from the onset and continued to increase throughout exercise. The total and total essential [AA] of muscle were significantly increased at exhaustion, whereas both the plasma and muscle branched-chain AA contents were unchanged. This suggests that protein catabolism was occurring during exercise and the branched-chain AA were used for energy and NH3 production.