Glial cells in the rat optic nerve were visualized by intracellular injections of horseradish peroxidase (HRP). A novel class of cell was encountered that was presumed to be an oligodendrocyte on the basis of arguments related to its light microscopic appearance after intracellular staining (Butt and Ransom, Glia 1989;2:470-475). These cells had 10-20 parallel processes 200-300 microns long that were oriented exclusively along the long axis of the optic nerve; the parallel processes were connected to the cell body by thin branches 15-30 microns long. To determine if these HRP-filled cells were oligodendrocytes, they were examined ultrastructurally; all cells examined in this way were unequivocally found to be myelin-producing oligodendrocytes. The oligodendrocytes contained intracellular organelles that were characteristic of this cell type, including abundant Golgi profiles and microtubules. In addition, HRP was found to fill the inner and outer tongue processes of myelin sheaths and the paranodal loops at nodes of Ranvier, proving that the entire cytoplasmic border surrounding the myelin sheath rapidly communicates by intracellular diffusion with the cell body. This electron microscopic study demonstrates that oligodendrocytes in the rat optic nerve can be positively identified by their distinctive light microscopic appearance after intracellular dye injection, and provides light microscopic criteria for establishing the number, distribution, and dimensions of the myelin segments provided by individual oligodendrocytes.