Abstract The ventral horn of the spinal cord is profusely innervated by serotonin (5-hydroxytryptamine, 5-HT) which presumably modulates locomotor activity through motoneurons. However, direct evidence of correlation between 5-HT release and activation of motoneurons is still lacking. In order to appreciate the functional characteristics of this innervation, we have used microdialysis to monitor the release of 5-HT in the spinal cord of rats spontaneously running on a treadmill. For this purpose, we developed an original surgical procedure adapted for the chronic implantation of a microdialysis probe in the lumbar spinal cord. The probe was kept in place for 40 days, and microdialysis experiments were carried out at days 8, 16, 20 and 32. 5-HT was detected with HPLC coupled to electrochemistry. This technique demonstrated that release of 5-HT is not increased during exercise. However, a significant decrease was measured during postexercise rest. 5-HT could still be detected 32 days after probe implantation. Detailed histological and immunocytochemical analysis based on glial fibrillary acidic protein and 5-HT immunocytochemistry showed minimal gliosis and the presence of serotonergic varicose fibers, respectively, at the probe contact. It thus appears that microdialysis can be performed through a probe implanted chronically in the spinal cord of unrestrained rat during an endurance running exercise.