Serotonergic projections from raphe nuclei arrive in the lumbar enlargement of the spinal cord during the late fetal period in the rat, a time window during which the locomotor-related left/right and flexor/extensor coordinations switch from synchrony to alternation. The goal of the present study was to investigate the role played by serotonin (5-HT) in modulating the left/right and flexor/extensor alternations. Fictive locomotion was induced by bath application of N-methyl-D,L-aspartate (NMA) in the in vitro neonatal rat spinal cord preparation. By means of cross-correlation analysis we demonstrate that 5-HT, when added to NMA, improves left/right and flexor/extensor (recorded from the 3rd and 5th lumbar ventral roots, respectively) alternations. This effect was partly reproduced by activation of 5-HT(2A/2C) receptors. We then tested the contribution of endogenous 5-HT to NMA-induced fictive locomotion. Reducing the functional importance of endogenous 5-HT, either by inhibiting its synthesis with daily injections of p-chloro-phenylalanine (PCPA), starting on the day of birth, or by application of ketanserin (a 5-HT(2) receptor antagonist) or SB269970 (a 5-HT(7) receptor antagonist), disorganized the NMA-induced locomotor pattern. This pattern was restored in PCPA-treated animals by adding 5-HT to the bath. Blocking 5-HT(7) receptors disorganized the locomotor-like rhythm even in the absence of electrical activity in the brain stem, suggesting that NMA applied to the spinal cord does not cause 5-HT release by activating a spino-raphe-spinal loop. These results demonstrate that 5-HT is critical in improving the locomotor-related alternations in the neonatal rat.