Various studies on isolated neonatal rat spinal cord have pointed to the predominant role played by the rostral lumbar area in the generation of locomotor activity. In the present study, the role of the various regions of the lumbar spinal cord in locomotor genesis was further examined using compartmentalization and transections of the cord. We report that the synaptic drive received by caudal motoneurons following N-methyl-d-l-aspartate (NMA)/5-HT superfusion on the entire lumbar cord is different from that triggered by the same compounds specifically applied on the rostral segments. These differences appear to be due to the direct action of NMA/5-HT on motoneuron membrane potential, rather than on premotoneuronal input activation. In order to assess the possible participation of the caudal lumbar segments in locomotor rhythm generation, the segments were over-stimulated with high concentrations of NMA or K+. We find that significant variations in motor cycle period occurred during the over-activation of the rostral segments. Over-activation of caudal segments only si+gnificantly increased the caudal ventral roots burst amplitude. We find that low 5-HT concentrations were unable to induce fictive locomotion under our experimental conditions. When a hemi-transection of the cord was performed between the L2-L3 segments, rhythmic bursting in the ipsilateral L5 disappeared while rhythmicity persisted on the contralateral side. Sectioning of the remaining L2-L3 side totally suppressed rhythmic activity in both L5 ventral roots. These results show that the thoracolumbar part of the cord constitutes the key area for locomotor pattern generation.