The earliest developing interneurons in the chick spinal cord can be divided into two groups: neurons in the ventral region whose axons pioneer the primitive longitudinal pathway (PL-cells) and neurons whose axons project circumferentially (C-cells) along the lateral marginal zone and join the ipsilateral or contralateral ventrolateral longitudinal pathways. To begin to examine the molecular cues for axonal pathway formation of these interneurons, we screened a variety of molecules from embryonic day (E) 2 to E6.5 [stage 14-30 of Hamburger and Hamilton (1951) J. Morphol. 88:49-92]. These include cell adhesion and related molecules (G4, F11, neurofascin, N-cadherin, TAG-1-like molecule), extracellular matrix (ECM) molecules (laminin, fibronectin, heparan sulfate proteoglycan, laminin-heparan sulfate proteoglycan complex, and collagen type IV), and receptors for ECM molecules (beta 1-class integrin). PL-cells first expressed neurofascin at stage 14+ before the onset of axonogenesis. When the PL-cells began to extend their axons at stage 15, they expressed G4 and avian TAG-1-like molecules, as well as neurofascin, on both cell bodies and longitudinal axons. In the following stages, PL-cells continued to strongly express neurofascin and G4 on their fasciculating axons, suggesting the involvement of these glycoproteins in growth and fasciculation. C-cells began to express G4 and TAG-1-like molecules on cell bodies and axons at stage 15-16 shortly after axonal growth. In the following stages, C-cells expressed several cell adhesion molecules differentially on their axonal segments. The proximal segment of C-axons in the circumferential pathway strongly expressed a TAG-1-like molecule, whereas the distal segment in the longitudinal pathway strongly expressed G4 and neurofascin. The commissural axonal segment in the floor plate expressed TAG-1-like molecule, neurofascin, N-cadherin, and beta 1-class integrin. The basement membrane around the spinal cord was enriched with ECM glycoproteins (laminin, fibronectin, heparan sulfate proteoglycan, and collagen type IV) during the stages examined (stage 15-27), and commissural C-cell axons became strongly integrin positive in the floor plate where they contacted the basement membrane. These data indicate that interneurons may use multiple molecules during axonal pathway formation, depending on cell type, pathway position, and developmental stage.