Development of neuronal circuits generating locomotor activity was studied using an isolated lumbar spinal cord preparation from fetal and neonatal rats. Bath application of N-methyl-D-aspartate (NMDA) or 5-HT evoked patterned motor activity resembling that seen during normal fictive locomotion on embryonic day (E) 20.5. Glycine-mediated inhibition was essential to the formation of this coordinated motor activity. In preparations from fetuses at the earlier stages (E14.5-E16.5), we observed spontaneous motoneuronal activity and chemically induced rhythmic bursts, which were synchronized on the two sides in the corresponding ventral roots. The spontaneous activity was not blocked by kynurenate, the glutamate receptor blocker, although it was completely abolished by strychnine, the glycine receptor antagonist. A brief application of glycine evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. It is concluded that glycine functions transiently as excitatory transmitters at these stages. These results suggest that functional change in glycine-induced responses during development plays an important role in differentiation of the neuronal circuits generating locomotion.