Aims: Muscle fatigue is a phenomenon we all experience in everyday life. It is characterized by a decrease of muscle force and induced by motor exercise. It has two components: one occurs in the muscle itself and the other in the central nervous system (both in the neocortex and in motoneurons). Many indirect evidences suggest that the central component of fatigue is induced by an increase in the concentration of serotonin (5-HT). For this reason, we investigated the cellular mechanisms that may link 5-HT to a decrease of motoneuronal activity. Methods: We tested if the activation of serotonergic receptors on adult motoneurons had an inhibitory effect. For this, we recorded motoneurons in a slice preparation of the turtle spinal cord, by means of the patch clamp technique. With an iontophoresis pipette, we applied 5-HT or 5-HT receptor agonists, on different compartments of the motoneuron. Results: The release of 5-HT at the axon initial segment (AIS) inhibited the genesis of action potentials. We showed that this effect was induced by the activation of 5-HT1A receptors that inhibit a sodium current. When tested in other compartments of the motoneuron (soma, dendrite, distal axon), 5-HT1A receptor agonist had no effect on sodium currents. This demonstrates that the activation of 5-HT1A receptors at the AIS inhibits the output of motoneurons. Conclusion: The release of 5-HT on motoneurons is correlated with the level of motor activity. Since no serotonergic synapse has been reported on the AIS, we suggest that during intense motor exercise, the 5-HT1A receptors at the AIS are activated by a spillover of 5-HT. This, in turn, inhibit the genesis of action potentials in motoneurons and hence the strength developed by the muscle. This finding is the first cellular mechanism that explains central fatigue.