Abstract We have prepared dissociated cell cultures from adult rat optic nerve. After 48 h in culture cells of both fibroblastic and process-bearing morphology could be recognised. Using indirect immunofluroescence and cell-type specific markers it was possible to unambiguously identify astrocytes and oligodendrocytes in culture. Approximately 0.1% of cells were galactocerebroside + ligodendrocytes, 20% were GFAP + astrocytes and 30% were fibronectin +, Thy-1 +, GFAP − fibroblastoid cells. Fifty percent of the GFAP + astrocytes expressed the Thy-1 antigen but none of them expresses fibronectin. All of the oligodendrocytes expressed intracellular myelin basic protein. Thus, glial cells isolated from adult tissues were similar in their antigenic properties to such cells isolated from neonatal tissues.