Three types of glial cells have been previously described in cultures of neonatal rat optic nerve--oligodendrocytes, type 1 astrocytes, and type 2 astrocytes--which can be distinguished using three different antibodies: antigalactocerebroside antibodies recognize oligodendrocytes; antibodies against glial fibrillary acidic protein recognize both types of astrocytes, while the A2B5 monoclonal antibody distinguishes between the two, binding to type 2 but not type 1 astrocytes. It was subsequently shown that oligodendrocytes and type 2 astrocytes, but not type 1 astrocytes, develop in cultures of 7 day optic nerve from a common, A2B5+ progenitor cell. In the present study, the distribution of rat neural antigen-2 (Ran-2), a cell-surface antigen defined by a monoclonal antibody, has been examined on optic nerve cells. It is demonstrated that, in contrast to A2B5, Ran-2 is present on type 1 but not type 2 astrocytes in optic nerve cultures. More importantly, it is shown that Ran-2 and A2B5 antibodies react with largely nonoverlapping populations of cells in cell suspensions of embryonic Day 17 (E17) and postnatal Day 1 (P1) optic nerve, and that the Ran-2+, A2B5- population contains type 1 astrocytes and their precursors while the A2B5+,Ran-2- population contains the progenitor cells for oligodendrocytes and type 2 astrocytes. These findings provide strong evidence that the glial cells of the rat optic nerve develop as two distinct lineages--one giving rise to type 1 astrocytes and the other to oligodendrocytes and type 2 astrocytes--and that the two lineages diverge as early as E17.