Serotonin (5-HT) can potently activate and modulate spinal locomotor circuits in a variety of species. Many of these findings have been obtained by applying serotonin exogenously to the isolated spinal cord of in vitro preparations, which has the drawback of indiscriminately activating extrasynaptic receptors and neurons. To investigate the role of endogenously released serotonin in modulating locomotor networks, the selective serotonin reuptake inhibitor citalopram was used. Fictive locomotion was elicited by either electrical stimulation of the brainstem or the sacral 4 (S4) dorsal root. The addition of 20 microm of citalopram caudal to thoracic segment 5 (T5) had an overall inhibitory effect on the lumbar central pattern generator (CPG). Left-right and flexor-extensor coupling were significantly decreased, and there was also a phase shift in the flexor-extensor relationship. In addition, there was a significant decrease in burst amplitude. These effects were observed during both afferent and brainstem evoked fictive locomotion. When citalopram was added in the presence of 5-HT(1A) and 5-HT(1B) antagonists, the inhibitory effects were largely reversed. The remaining excitatory effects were mediated by 5-HT(7) and 5-HT(2) receptors. These results suggest that endogenous 5-HT release can modulate locomotor-like activity early in neonatal development.