The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.