We evaluated whether peripheral ammonia production during prolonged exercise enhances the uptake and subsequent accumulation of ammonia within the brain. Two studies determined the cerebral uptake of ammonia (arterial and jugular venous blood sampling combined with Kety-Schmidt-determined cerebral blood flow; n = 5) and the ammonia concentration in the cerebrospinal fluid (CSF; n = 8) at rest and immediately following prolonged exercise either with or without glucose supplementation. There was a net balance of ammonia across the brain at rest and at 30 min of exercise, whereas 3 h of exercise elicited an uptake of 3.7 ± 1.3 µmol min^-1 (mean ± S.E.M.) in the placebo trial and 2.5 ± 1.0 µmol min^-1 in the glucose trial (P < 0.05 compared to rest, not different across trials). At rest, CSF ammonia was below the detection limit of 2 µM in all subjects, but it increased to 5.3 ± 1.1 µM following exercise with glucose, and further to 16.1 ± 3.3 µM after the placebo trial (P < 0.05). Correlations were established between both the cerebral uptake (r² = 0.87; P < 0.05) and the CSF concentration (r² = 0.72; P < 0.05) and the arterial ammonia level and, in addition, a weaker correlation (r² = 0.37; P < 0.05) was established between perceived exertion and CSF ammonia at the end of exercise. The results let us suggest that during prolonged exercise the cerebral uptake and accumulation of ammonia may provoke fatigue, e.g. by affecting neurotransmitter metabolism.