Neurons with axons that extend to the contralateral side of the spinal cord--commissural interneurons (CINs)--coordinate left/right alternation during locomotion. Little is known about the organization of CINs in the mammalian spinal cord. To determine the numbers, distribution, dendritic morphologies, axonal trajectories, and termination patterns of CINs located in the lumbar spinal cord of the neonatal rat, several different retrograde and anterograde axonal tracing paradigms were performed with fluorescent dextran amines and the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). CINs with ascending (aCINs) and descending (dCINs) axons were labeled independently. The aCINs and dCINs occupied different but overlapping domains within the transverse plane. The aCINs were clustered into four recognizable groups, and the dCINs were clustered into two recognizable groups. All dCINs and most aCINs were located within the gray matter, with somata ranging from 10-30 microm in diameter and with large, multipolar dendritic trees. One group of aCINs was located outside the gray matter along the dorsal and dorsolateral margin and had dendrites that were nearly confined to the dorsolateral surface. All CIN axons traversed the ventral commissure at right angles to the midline. CIN axons coursed up to six or seven segments rostrally and/or caudally in the ventral and ventrolateral white matter and gave off collaterals over a shorter range, predominantly to the ventral gray matter. These findings show that the lumbar spinal cord of the neonatal rat contains substantial numbers of CINs with axon projections and collateral ranges spanning several segments and that CINs projecting rostrally vs. caudally have different distributions in the transverse plane. The study provides an anatomical framework for future electrophysiological studies of the spinal neuronal circuits underlying locomotion in mammals.