Using immunocytochemical methods, the cell adhesion molecule L1 was detected on axons crossing in the dorsal commissure of developing rat spinal cord. Immunoreactive axons were found in locations similar to fiber bundles illustrated by Ramón y Cajal and designated the anterior, middle and posterior bundles of the dorsal commissure. L1-immunoreactive dorsal commissural axons were first observed on embryonic day 17 (E17), appeared more numerous by E19, and remained detectable in early postnatal ages. The massive middle axon bundles extended bilaterally from the dorsolateral funiculi towards the midline and crossed in the central part of the commissure. In horizontal sections, bundles of L1-labeled middle axons were observed to traverse the dorsal commissure in a periodic pattern along the entire rostrocaudal extent of the spinal cord. Bundles of glutamic acid decarboxylase (GAD65)-positive axons were detected crossing in the middle and posterior regions of the dorsal commissure between E17 and E20. Results from double-labeling experiments demonstrated that GAD65-positive fibers were embedded in larger bundles of L1-labeled axons and that some dorsal commissural axons were double-labeled. To determine if there were axons crossing in the dorsal commissure that did not express L1, double-labeling experiments were conducted using neurofilament and L1 antibodies. Results indicated that bundles of axons identified with anti-neurofilament antibodies were also L1-positive, whereas individually coursing axons within the commissure were L1-negative. The predominance of L1 on fiber bundles traversing the dorsal commissure adds to the growing evidence that this molecule may play a role in axon outgrowth and fasciculation.