Primary cultures of cerebral glia derived from neonatal rat brain were utilized to determine 1) the developmental changes of dolichol-linked oligosaccharide and N-linked glycoprotein biosyntheses, 2) the enzymatic correlates of these developmental changes, and 3) the temporal relations between the biosyntheses of dolichol-oligosaccharide and N-linked glycoproteins and the biochemical expression of astrocytic and oligodendroglial differentiation. Marked, parallel developmental increases in the rates of incorporation of [3H]glucosamine into both dolichol-oligosaccharide and glycoprotein were observed, with maximal rates achieved after 9-10 days in culture and little change occurring over the next 10 days in culture. Concerning the enzymatic correlates, dolichol kinase exhibited a moderate developmental increase with a maximum at 5 days in culture, whereas the activities of the three critical enzymes that utilize dolichyl phosphate in the synthesis of the dolichol-linked oligosaccharide, i.e., N-acetylglucosaminylphosphotransferase (GlcNAc-1-P transferase), mannosylphosphoryldolichol (Man-P-Dol) synthase, and glucosylphosphoryldolichol (Glc-P-Dol) synthase, reached maxima after 6-9 days in culture. Both the activity of Man-P-Do1 synthase in vitro and the rate of formation of its product, Man-P-Dol, in intact cells were shown to correlate closely with the rates of oligosaccharide and glycoprotein biosyntheses. An important regulatory role for Man-P-Do1 synthase and its product, Man-P-Dol, was suggested further by the demonstration of a maturationdependent activation by Man-P-Do1 of GlcNAc-1-P transferase, the first committed step in the pathway. Two enzymatic markers of astrocytic (glutamine synthetase) and oligodendroglial (2' ,3'-cyclic nucleotide 3'-phosphohydrolase) differentiation exhibited marked developmental increases in activity with onset at the time of attainment of peak rates of dolichololigosaccharide and glycoprotein biosyntheses. Importance of the latter processes for glial differentiation is suggested.