This paper describes the differential expression and localization of myelin components within membrane sheets produced by oligodendrocytes in vitro. In double‐labeling experiments using antibodies to the myelin antigens 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and galactocerebroside (GC), the two antigens were coexpressed in at least 95% of oligodendrocytes at all ages examined. A small population of relatively undifferentiated cells expressed one antigen before the other. Within the membrane sheets produced by the cultured cells, CNP and GC are distributed differently. CNP is highly concentrated in cell bodies, in a network of processes extending from the cell body into the sheets, and around the perimeter of the sheets. CNP staining cannot be detected in some areas within the body of the sheet. When present, it is of low intensity. Under our labeling conditions, GC staining is found throughout the membrane sheets, except in the network of veins which are CNP+. GC and myelin basic protein (MBP) staining are seen in similar membrane domains even though GC is a surface component while MBP resides on the cytoplasmic face.

Both the timing and localization of CNP immunostaining show that CNP is as early a marker for oligodendrocytes as GC, and support the idea that CNP may play a structural role in the myelin membrane. Double‐labeling studies with GC and CNP antibodies also show that the true shape of a cell and the extent of its development are not always revealed by a single antigen. The differential distribution of antigens within membrane sheets illustrates that they contain areas of structural specialization that may reflect the situation in intact myelin.