The distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating oligodendrocytes of primary and secondary mouse brain cell cultures by single- and double-label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet-like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39-41 days, harvested by scraping, enriched for oligodendrocytes, and plated at low cell density. After 1 week, isolated oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine-grained patches of MBP, which were surrounded by networks of MBP- processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP are differentially located in cultured mouse oligodendrocytes. Furthermore, the precise distribution of these myelin-specific antigens is dependent on culture conditions.