The node of Ranvier is a complex macromolecular assembly of ion channels and other proteins that is specialized for the rapid propagation of the action potential. A full understanding of the processes responsible for the assembly and maintenance of the node requires first the identification and characterization of the proteins found there. Here we show that NG2, a structurally unique chondroitin sulfate proteoglycan, is a molecular component of the node of Ranvier in the peripheral nervous system. In adult sciatic nerve, NG2 is (1) associated with thin, elongated fibroblast-like cells, (2) on some but not all basal laminae, and (3) at nodes of Ranvier. At the nodes, NG2 is restricted to the nodal gap and is absent from the paranodal or juxtaparanodal region. In dissociated cell cultures of adult sciatic nerve, perineurial fibroblasts but not Schwann cells express NG2 on their surfaces. Approximately 45% of the total NG2 in peripheral nerves is in a soluble, rather than particulate, subcellular compartment. NG2 is also present in membrane fractions that also contain high levels of voltage-dependent sodium channels, caspr, and neuron-glia related cell adhesion molecule. These medium-density membranes likely correspond to the nodal and paranodal region of the axon-Schwann cell unit. These results suggest a model in which perineurial fibroblasts secrete or shed NG2, which subsequently associates with nodes of Ranvier. The growth-inhibitory and anti-adhesive properties of NG2 may limit the lateral extension of myelinating Schwann cells as nodes mature. NG2 may also participate in the barrier functions of the perineurial linings of the nerve.