The expression and regulation of nestin, an intermediate filament protein associated with neuroepithelium-derived progenitor cells, was studied in developing oligodendrocytes. We analyzed glial cells cultured from late embryonic rat cerebral cortex, tissue prints from neonatal rat brains, and the oligodendrocyte cell line CG-4. Using cortical- or CG-4-derived oligodendrocyte lineage cells, Northern blot analysis demonstrated that nestin mRNA was highly expressed in proliferating O-2A progenitors, but downregulated in differentiated oligodendrocytes. Immunocytochemistry of cultured cells and tissue prints demonstrated that pre-O-2A cells and O-2A progenitors expressed high levels of the nestin protein. In contrast, nestin expression decreased during the pro-oligodendroblast stage, and the majority of oligodendrocytes were not labeled with anti-nestin antibodies. Culture conditions (PDGF+bFGF or B104-conditioned medium) that maintained O-2A progenitor proliferation and prevented differentiation enhanced expression of nestin mRNA transcripts and protein. In comparison, analysis of cortical astrocyte cultures demonstrated that type 1 astrocytes, but not type 2, expressed nestin mRNA and protein. Our findings demonstrate that nestin is expressed in developing glial cells, which is suggestive that this intermediate filament protein may play an important role during gliogenesis.