Assembly of extracellular collagen fibrils and Schwann cell basal lamina has previously been identified as a prerequisite for compact myelin formation in the peripheral nervous system. Synthesis of this extracellular matrix (ECM) in vitro required the presence of serum and ascorbic acid. Using rat embryonic dorsal root ganglion neurons and Schwann cells, we have developed a fully defined medium in which myelination occurs. In the absence of ascorbic acid, normal myelin was formed without ECM assembly. This demonstrates that although myelination and ECM assembly are usually closely linked, ECM formation is not a prerequisite for myelination in vitro.