Axon growth in vitro may be inhibited by contact with oligodendrocytes, but most axons grow readily on the surface of astrocyte monolayers. Since both cell types are in close contact with one another in the damaged nervous system, we have examined the growth of axons on cultures which contain both astrocytes and oligodendrocytes. Cultures derived from neonatal rat forebrain develop with a monolayer of large flat astrocytes attached to the culture dish, and with many smaller cells of the oligodendrocyte lineage on their surface. Dorsal root ganglia placed on these cultures grow axons readily, the overall extent of growth being unaffected by the presence or absence of oligodendrocytes, many of which express galactocerebroside and the inhibitory molecule janusin. A previous set of experiments had shown that growth of these axons is inhibited by oligodendrocytes by themselves. Scanning electron microscopy coupled with silver-intensified immunostaining reveals that the axons grow on the surface of the astrocytic layer, underneath the oligodendrocytes, and are therefore in contact with both cell types as they grow. The presence of astrocytes therefore alters the results of axonal contact with oligodendrocytes.