The Ig cell adhesion molecules (CAM) neurofascin (NF) and Nr-CAM are localized at developing nodes of Ranvier in peripheral myelinated axons prior to clustering of Na+ channels. Different isoforms of NF are expressed on neurons and glia, and NF binding on both cells has been suggested to play roles in node and paranode formation. To clarify the role of NF further, we analyzed effects of NF-Fc fusion proteins in Schwann cell-DRG neuron myelinating cocultures. NF-Fc significantly inhibited nodal clustering of Na+ channels, ankyrin G, and betaIV spectrin, and modestly reduced Caspr clustering at paranodal junctions; it did not significantly affect lengths or numbers of myelin-positive segments, axon initial segments, or accumulations of phosphorylated-ERM proteins in Schwann cell nodal microvilli. NF-Fc binds to Schwann cells but little or no binding to DRG neurons was detected. The results suggest a critical early role for axonal NF in clustering of Na+ channels at nodes of Ranvier via interactions with receptors on Schwann cells.