The aim of this study was to investigate if enhanced peripheral ammonia production during exhaustive exercise increases ammonia detoxication in brain mediated by glutamine synthesis, and subsequently influences glutamate and gamma-aminobutyric acid (GABA) levels. This neurotransmitter production is related to the metabolism of glutamine. A group of rats was trained for 6 weeks by treadmill running (TR). They were compared to a group of untrained rats (UN). At the end of training, half of TR and UN rats were submitted to one session of treadmill running until exhaustion (288+/-12 min and 62+/-5 min in TR and UN group, respectively). At exhaustion, running and control rats were sacrificed in order to collect blood and to take samples of the following brain structures: cortex, striatum and cerebellum. Treadmill running until exhaustion induced an increase in blood ammonia by 140% without significant differences between TR and UN groups. Brain ammonia increased in both groups. However, TR group exhibited values 50% higher than those observed in UN group. Brain glutamine was increased at exhaustion in all groups of running rats by 30-75% of basal value whereas the glutamate only decreased in TR rats which were able to run for a longer time. In this group, the GABA level decreased in striatum. These data confirm that enhanced brain ammonia level during exercise stimulates glutamine synthesis as a mechanism of detoxication. After several hours of running, a reduction in brain glutamate levels was observed in all brain structures in trained rats but only in the striatum in untrained animals. The reduced availability of this GABA precursor decreases GABA levels only in the striatum of TR group by 45% of the resting value. These results suggest a relation between cerebral changes in neurotransmitters and excitatory amino acids, such as glutamate and GABA, and central fatigue.