Excitatory amino acids are known to activate the spinal neural network that organize locomotor activity in various species. In this study, the role of various compounds which alter the functioning of the N-methyl-D-aspartate receptor (glycine, Mg2+ ions and spermine) was investigated during fictive locomotion, using an in vitro isolated spinal cord preparation from neonatal rats. Locomotor-like activity induced by excitatory amino acids was recorded both extra- and intracellularly. 7-chloro-kynurenic acid, an antagonist of the glycine site at the N-methyl-D-aspartate receptor, depressed the N-methyl-D-aspartate component of the synaptic inputs received by the motoneurons. Glycine at low concentrations had no effect on locomotor activity, while 7-chlorokynurenic acid increased the locomotor period and decreased the burst amplitude in a dose-dependent manner. Removal of Mg2+ ions from the saline facilitated the N-methyl-D-aspartate-mediated response, and triggered spontaneous bursting activity, abolished by 2-amino-5-phosphonovaleric acid, an antagonist of the N-methyl-D-aspartate receptor. The polyamine, spermine, did not change the locomotor parameters. On the contrary, arcaine, a putative antagonist of the polyamine site on the N-methyl-D-aspartate receptor, increased locomotor activity. The effects of arcaine were counteracted by spermine. These results suggest that glycine and spermine are present at saturating concentrations on the N-methyl-D-aspartate receptor during ongoing locomotion. Together with Mg2+ ions, these endogenous regulators contribute to control the level of activity of the N-methyl-D-aspartate receptor in the spinal cord of the neonatal rat.