Axons undergo substantial changes in radial growth during the course of development. Recent evidence suggests that axonal diameter may be controlled by the state of neurofilament (NF) phosphorylation. Using dorsal root ganglion (DRG)-Schwann cell co-cultures, we provide direct evidence that phosphorylation of NF is regulated by myelination. NF phosphorylation increased upon myelination of DRG neurons by Schwann cells. The increase in NF phosphorylation was reflected both as an increase in immunoreactivity with the antibody SMI31, specific for phosphorylation-dependent NF epitopes, and a concomitant decrease in immunoreactivity with SMI32, specific for nonphosphorylated NF epitopes. The increase in NF phosphorylation induced by myelination in the neuron-glia co-cultures was similar to NF phosphorylation seen in sciatic nerve extracts of mice with normal myelination compared to Trembler J mouse littermates in which myelination of peripheral nerves is compromised. Using an in situ gel kinase assay, we have detected changes in individual NF kinase activities during myelination. In particular, a 35-kDa kinase activity was induced by myelination, whereas a 42-kDa kinase decreased in activity. We discuss the possibility that these and other kinases may be involved in signaling processes between neurons and glia during myelination.