Some macroglial cells of the O-2A lineage express glutamate receptor channels of the quisqualate/kainate type and take up extracellular cobalt when activated by glutamate agonists. These cells can be identified both in vitro and in situ following precipitation and intensification of the intracellular cobalt. We have used this technique to characterize these cells in the developing and adult rat optic nerve. In purified cultures of optic nerve cells, O-2A progenitor cells and type 2 astrocytes took up cobalt in the presence of quisqualate, while oligodendrocytes, type 1 astrocytes, and microglial cells did not. When whole optic nerves of various postnatal ages were exposed to quisqualate and cobalt, a subpopulation of glial cells took up cobalt. Cobalt uptake in vitro and in situ was blocked by 6-cyano-7- nitroquinoxaline-2,3-dione. The number, morphology, and spatial distribution of cobalt-filled cells in situ varied with age. In perinatal nerves, 9% of glial cells took up cobalt. These cells had a simple unipolar or bipolar morphology and were two to three times more concentrated at the chiasm end than at the eye end of the nerve. During subsequent development, this gradient disappeared and the cobalt-filled cells became progressively more complex in morphology and increased in number and density, reaching a peak toward the end of the second postnatal week. The number subsequently declined to about 16,000 (7%) in the adult nerve. The processes of some cobalt-filled cells appeared to contact nodes of Ranvier. All cobalt-filled cells in 2 1/2-week-old optic nerves had a similar ultrastructural appearance and did not resemble either mature oligodendrocytes or astrocytes. Our results suggest that the cells stimulated by quisqualate to take up cobalt in the optic nerve are the in vivo counterpart of O-2A progenitor cells. We found no evidence that any of these cells are type 2 astrocytes.