We describe a simple, rapid, and efficient method, based on separation on a Percoll centrifugation gradient, to purify glial progenitor cells from newborn rat brains. Cytofluorimetry analysis of the isolated cell population showed that 75 +/- 8 and 86 +/- 7% of the cells were A2B5- and R24-positive, respectively. Transmission electron microscopy examination of the purified cell population confirmed their homogeneity and illustrated their typical morphology, as previously described in situ. Assay of UDP-galactose-ceramide galactosyltransferase, 3'-phosphoadenosine 5'-phosphosulfate galactosylceramide sulfotransferase, and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities showed that the levels of these enzymes were 446, 76, and 11 times lower, respectively, than the levels measured in mature oligodendrocytes. Low levels of mRNA coding for 2',3'-cyclic nucleotide 3'-phosphohydrolase and myelin proteolipid protein, but not for myelin basic protein, were present in the glial progenitor cells. At the time of isolation, 40% of the cells in the population were dividing, and the cells could easily be expanded in culture. After 3 weeks of culture in the presence of 1% fetal calf serum, 75% of the cells had differentiated into galactosylceramide-positive oligodendrocytes. When the culture took place in the presence of 10% fetal calf serum, only 2% of the cells expressed galactosylceramide, and 60% were glial fibrillary acidic protein-positive astrocytes; half of them were also A2B5 positive.