A direct role for neurons in CNS myelination has yet to be demonstrated. CNS myelination can be examined in cerebellar slice cultures, which faithfully reproduce both synthesis and wrapping of myelin. In an attempt to demonstrate a role for axolemma in this process, we generated more than 2000 axolemma-reactive monoclonal antibodies. One clone, G21.3, repeatedly blocked myelination in cerebellar slices, as documented by both biochemistry and morphology. The antibody caused a dramatic reduction in myelin lipid and protein synthesis. CNS white matter, sciatic nerve, and neuronal cultures were positively stained with G21.3, whereas oligodendrocytes and myelin were fully negative. The antibody identified a restricted number of proteins in purified axolemma. These results suggest a direct involvement of axons in CNS myelination.