The ventrolateral funiculus (VLF) in the spinal cord contains important ascending and descending pathways related to locomotion and interlimb coordination. The primary purpose of this descriptive study was to investigate the distribution of inter-enlargement pathways in the adult rat spinal cord with an emphasis on the VLF. We made discrete unilateral injections of Fluoro-Gold (FG) into the right VLF at thoracic segment (T) 9, and either unilateral or bilateral injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5-6, C7-8, or lumbar (L) 2 segmental levels. Inter-enlargement neurons with ascending axons in the right VLF were found bilaterally in laminae VII and VIII throughout the rostral lumbar spinal cord (L1-L3) and predominantly contralaterally in the caudal lumbosacral (L4-S1) spinal cord. Following left unilateral FR injections at C5-6 or C7-8 and right unilateral VLF injections of FG at T9, very few double-labeled neurons could be found anywhere in the lumbar spinal cord. Similar injections of FR at L2 revealed an almost symmetrical bilateral distribution of double-labeled neurons throughout the cervical spinal cord (C1-8). These results describe ascending and descending pathways within the spinal cord that interconnect the two enlargements and involve both commissural and ipsilateral interneurons. The majority of inter-enlargement neurons had axons within the VLF at T9. These observations support the hypothesis that the VLF contains long ascending and descending axons with propriospinal inter-enlargement, commissural and ipsilateral connections that are anatomically well-suited to mediate interlimb coordination. © 2006 IBRO.