To elucidate the role of neuron-glia cell adhesion molecule (Ng-CAM) in axonal pathway formation of avian spinal interneurons, we have examined the ultrastructural expression of Ng-CAM in the developing spinal cord, by using a preembedding immunocytochemical method. Ng-CAM immunoreactivity was punctate and was restricted to cell surfaces. In accordance with our previous light microscopic observations (Shiga et al., '90), the earliest developing spinal interneurons were Ng-CAM-positive on their cell bodies, axons, and growth cones. Axons and growth cones that were either fasciculated or in contact with each other strongly expressed Ng-CAM, thus indicating the possible involvement of Ng-CAM in fasciculation of axons and in the contact guidance of growth cones along preexisting axons. By using higher resolution immunoelectron microscopy, the present study has also revealed new information on the subcellular localization of Ng-CAM on developing spinal interneurons, neuroepithelial cells, and floor plate cells. Although Ng-CAM immunoreactivity was prominent on both axons and growth cones, these structures were Ng-CAM-negative when they contacted the basal lamina around the spinal cord. By contrast, Ng-CAM was detectable on the surface of both neuroepithelial cells and floor plate cells only when they made contact with the Ng-CAM-positive axons and growth cones of interneurons. These results suggest that the subcellular distribution of Ng-CAM is regulated differentially, depending on the apposing cell surfaces, and that such differential and developmentally regulated expression may contribute to the elongation, fasciculation, and guidance of spinal axons.