The aim of the present study was to investigate the effects of physical exercise (running) on serotonin (5-hydroxytryptamine, 5-HT) synthesis and metabolism in midbrain on the one hand, and in striatum and hippocampus on the other hand. To address such a question, tryptophan (TRP) and 5-hydroxytryptophan (5-HTP) were measured in running rats pretreated with an inhibitor of aromatic amino acid decarboxylase, namely NSD 1015. In another series of experiments, the consequences of a TRP load on TRP, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were compared in resting and running rats. Although running triggered a 30% increase in TRP levels in the three brain regions examined, inhibition of 5-HT synthesis by NSD 1015 was found to promote increased (midbrain), unchanged (striatum) or decreased (hippocampus) 5-HTP accumulation in the running situation, respectively compared to that measured in the resting situation. Inasmuch as running-induced elevation in TRP was not associated with an increased 5-HTP accumulation in the striatum and the hippocampus, the consequences of running on regional TRP, 5-HT and 5-HIAA levels were analyzed in saline- and TRP-injected rats. Indeed, running, per se, was found to increase central TRP, 5-HT and 5-HIAA levels. On the other hand, a TRP load that promoted identical increases in central TRP levels in running and resting rats revealed that running, according to the region examined, differentially affected TRP utilization in the 5-HT synthesis pathway. Thus, in the midbrains of the resting and running rats, respective 210-250% increases in TRP led to identical 25% increases in 5-HT and 90% increases in 5-HIAA levels. Conversely, in hippocampus, TRP loads triggered marked increases in TRP levels that were similar in the controls and the runners, but the rise in 5-HIAA promoted by such a precursor load was found to be significantly minored in the runners, compared to the resting rats. Moreover, such a running-induced impairment in 5-HT synthesis and metabolism was even more observable in the striatum; thus, TRP loads which promoted identical increases in striatal TRP levels in the resting and the running rats respectively triggered a 50% and a 32% increase in 5-HT levels and a 76% and a 47% increase in 5-HIAA levels. The results presented herein indicate that under certain pharmacological conditions, TRP utilization into the 5-HT synthesis pathway is altered in serotonergic nerve terminals, but not in the cell bodies of the running rat.