Myelination was studied in aggregating cell cultures derived from mechanically 15- to 16-day fetal rat brains. Myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) were localized immunocytochemically in 20-micrometers-thick Vibratome and 1-micrometer- thick Epon sections at 15, 20, 25, and 30 days in vitro. The occurrence of these proteins was correlated with the ultrastructural appearance of oligodendrocytes and myelin sheaths and with biochemical levels of MBP, MAG, and the myelin-related enzyme, 2′,3′-cyclic nucleotide 3′- phosphodiesterase (CNP). MBP appeared in ultrastructurally immature oligodendrocyte cytoplasm at 15 days in vitro. As oligodendrocytes developed a more differentiated fine structure, MBP and MAG antisera stained oligodendrocyte processes and myelin sheaths. Immunostaining in Vibratome sections demonstrated that MBP was detectable in oligodendrocytes and myelin prior to MAG. At 25 days in vitro, all Vibratome sections contained MBP- and MAG-stained oligodendrocytes and myelin sheaths. Radioimmunoassays for MBP and MAG and enzyme assays for CNP in whole homogenates of the aggregates revealed that each of these components increased with the progression of myelination. However, MBP only reached 8% of the level in adult rat brain, while MAG and CNP increased to more than half of the adult level. The protein composition of myelin purified from 30-day aggregates resembled that of myelin purified from immature rat brain.