The pattern of expression of the neuron-glia cell adhesion molecule (Ng-CAM) among the presumptive intersegmental interneurons in the early chick embryo spinal cord (stage 14-19) was examined using whole-mount and cryostat preparations double-stained with anti-Ng-CAM antibody and a neuron-specific anti-beta-tubulin antibody. In brachial segments, primitive longitudinal cells (PL-cells), located in the ventrolateral region of the spinal cord, express Ng-CAM both on their cell bodies and longitudinally growing axons, beginning at Hamburger-Hamilton stage 15 (embryonic day (E) 2.5). Shortly thereafter, at stage 16, circumferential cells (C-cells), composed of associational and commissural interneurons in the dorsolateral and lateral region of the spinal cord begin to express Ng-CAM both on their cell bodies and their axons which project ventrally along the lateral surface of the spinal cord. In the following stages. PL-cells express Ng-CAM strongly and begin to fasciculate to form a longitudinal fascicle in the ventrolateral marginal region of the spinal cord. C-Cells either extend their Ng-CAM-positive axons ventrally without fasciculation to join the Ng-CAM positive ipsilateral longitudinal fascicle at stage 17 (which has already been formed by PL-cell axons), or they cross the midline to join the Ng-CAM positive contralateral longitudinal fascicle after growing through the floor plate at stage 19. By stage 19, cell bodies and proximal segments of the C-cell axons exhibit decreased Ng-CAM expression, whereas the distal segment of these axons, including growth cones, strongly express Ng-CAM. Many contacts were observed between Ng-CAM-positive growth cones and axons in the ventral region of the spinal cord, both within the longitudinal fascicle and the floor plate region, suggesting the involvement of Ng-CAM in neurite-neurite interactions.(ABSTRACT TRUNCATED AT 250 WORDS)