We report the study of the in vivo morphology, differentiation, and proliferation of oligodendrocytes (OLs) and their progenitors identified by the antiglycolipid antibodies O4, R-mAb, and O1 in postnatal rat cerebellum, using a novel immunocytochemical staining protocol which allows the analysis of the expression of OL-specific glycolipids in live, unfixed brain slices. An analysis of the individual cells identified in double label immunocytochemistry indicated that the order of antigen expression in OLs during in vivo development is, first, antigens recognized by O4, second, antigens recognized R-mAb, and third, antigens recognized by O1. This order of antigen expression is correlated with increasing morphological complexity and is a pattern mimicked in many culture systems. In vivo O4 identified 3 distinct stages of the OL lineage: (1) morphologically simple proligodendrocyte antigen+ (POA+) R-mAb- blast cells localized at the leading edge of myelinogenesis; (2) morphologically more complex R-mAb+O1- cells; and (3) actively myelinating O1+ [i.e., galactocerebroside+ (GalC)] OLs residing within the white matter. Only the POA+R-mAb- cells incorporated BrdU in animals that were prelabeled 3 hr before immunocytochemistry. We have demonstrated in vivo the subdivision of pre-GalC+ OL progenitors into shorter, biologically noteworthy, stages of maturation. A spatial comparison of the cell populations identified by O4, R-mAb, and O1 demonstrated a progressive wave of OL maturation from the base of the cerebellum toward the folia. The data are consistent with the hypothesis that multiprocessed O4+GalC- progenitors are the most mature stage of the OL lineage with significant proliferative capacity and the first postmigratory stage in normal development.