Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, 01, 04 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. 01 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. 04 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with 04 that had not yet begun to express the 01 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in 01-positive cells. Therefore, 04 stains, in addition to sulfatide and seminolipid, an unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing 04+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with 01, and thus R-mAb transiently stained a larger population of oligodendrocytes than did 01. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that 04, R-mAb, and 01 have multiple overlapping specificities, including previously unrecognized cross-reactions.