The purpose of this study was to characterize the selenium requirement for the normal differentiation of oligodendrocyte lineage cells. In primary mixed glial cultures prepared from newborn rat brains, the overall growth of cultures, as seen from the total RNA yield, was not significantly affected by selenium. However, 30 nM selenium was required for the normal upregulation the proteolipid protein, basic protein, and myelin-associated glycoprotein gene expression assessed by Northern blot analysis. Selenium deprivation during initial, rapid phase of the gene upregulation irreversibly suppressed the genes, indicating the existence of a critical period in oligodendrocyte differentiation. In purified oligodendrocyte cultures prepared by mechanical dislodging of progenitor (O-2A) cells from mixed glial cultures, total cell number and total RNA yield were virtually unaffected by selenium deprivation; however, the developmental upregulation of the myelin genes was profoundly attenuated. Immunocytochemical analysis confirmed the suppressive effect of selenium deficiency on the differentiation of oligodendrocyte lineage cells, as seen from a significant decrease in the population of GalC+ and O4+ cells. Because the number of GC+ cells was more reduced than the number of O4+ cells, the results indicate that selenium deficiency may specifically inhibit the progression from immature to mature oligodendrocytes.