Enzymatically dissociated cell suspensions from adult rat spinal cord were added at low densities (5 X 10(3) cells/culture) to cultures of pure dorsal root ganglion neurons. Oligodendrocytes, identified by immunostaining with a monoclonal antibody to galactocerebroside, began to proliferate by 4 days after their addition, forming large colonies of cells by the 14th day. Myelin formation by oligodendrocytes began 4 weeks after addition and myelin was abundant by 6 weeks. Oligodendrocyte proliferation and myelination did not require the immediate presence of astrocytes; the number of astrocytes overall remained low throughout the culture period. Preliminary studies indicated that the specific removal of galactocerebroside-positive cells from the cultures with anti-galactocerebroside antibody and complement 3 days after their addition prevented the subsequent generation of new oligodendrocytes and myelination. These preliminary results suggest that a major source of new myelinating cells in the adult central nervous system (CNS) might be already committed, galactocerebroside-positive, oligodendrocytes rather than uncommitted stem cells. The absence of cellular barriers between the myelinating cells and the medium make these cultures well suited for studies probing cellular and molecular mechanisms of myelination in the CNS.