Several experiments have demonstrated that rostral segments of the vertebrate lumbar spinal cord produce a rhythmic motor output more readily and of better quality than caudal segments. Here we examine how this rostrocaudal gradient of rhythmogenic capability is reflected in the spike activity of neurons in the rostral (L(2)) and caudal (L(5)) lumbar spinal cord of the neonatal rat. The spike activity of interneurons in the ventromedial cord, a region necessary for the production of locomotion, was recorded intracellularly with patch electrodes and extracellularly with tetrodes during pharmacologically induced locomotion. Both L(2) and L(5) neurons tended to be active in phase with their homologous ventral root. L(5) neurons, however, had a wider distribution of their preferred phases of activity throughout the locomotor cycle than L(2) neurons. The strength of modulation of the activity of individual L(2) neurons was also larger than that of L(5) neurons. These differences resulted in a stronger rhythmic signal from the L(2) neuronal population than from the L(5) population. These results demonstrate that the rhythmogenic capability of each spinal segment was reflected in the activity of interneurons located in the same segment. In addition to paralleling the rostrocaudal gradient of rhythmogenic capability, these results further suggest a colocalization of motoneurons and their associated interneurons involved in the production of locomotion.