Oligodendrocytes are target cells in the pathogenesis of multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS). During the course of the disease, inflammatory mediators may damage oligodendrocytes and their myelin sheaths. Differentiation of oligodendrocyte progenitors is an important step in the process of remyelination. In the present study, OLN-93 differentiation was studied in co-culture with C6 astrocytes as a natural source of growth and differentiation factors as well as after exposure to insulin-like growth factor-I (IGF-I). Morphological evaluation showed an increased degree of differentiation of OLN-93 cells after IGF-I administration, but not after co-culture with astrocytes. During early differentiation, 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and zonula occludens-1 (ZO-1) tight junction protein expression were significantly increased. However, neither astrocyte co-culture nor exposure to IGF-I further increased the expression of these markers. Although reverse transcriptase-polymerase chain reaction revealed myelin basic protein (MBP) mRNA expression not to be affected during differentiation, we did find increased MBP protein expression by Western blotting. ZO-1 protein and DM20 mRNA levels were increased during the course of differentiation and after IGF-I administration. The present findings suggest that ZO-1 may be used as a marker for OLN-93 oligodendroglia differentiation.