The developmental changes of dolichol kinase activity and dolichyl phosphate levels have been studied in rat brain. Because both dolichol kinase activity and dolichyl phosphate were enriched in microsomes, detailed study of this subcellular fraction was carried out. Dolichol kinase specific activity in brain microsomes increased postnatally 3-fold to a maximum at ca. 30 days of age. This increase was observed whether exogenous dolichol was present or not and whether Zn2+ or Ca2+ was utilized as the cation for the enzyme. Zn2+ was the most effective cation in developing brain, as we have shown previously for adult brain (Sakakihara, Y. and Volpe, J.J., J. Biol. Chem., 260 (1985) 15413-15419). Although the Vmax for the enzyme increased by three-fold with development, the Km for dolichol and for CTP did not change, indicating that the developmental increase was not related to an alteration in catalytic efficiency of the enzyme. A striking and parallel increase in dolichyl phosphate levels in brain microsomes was defined with development. Levels were lowest in one-day-old animals and then increased ca. 13-fold to a maximum at 30 days of postnatal age. The parallel increase in dolichol kinase activity and dolichyl phosphate levels in microsomes of developing brain suggests that dolichol kinase is the principal determinant of cellular levels of dolichyl phosphate, the critical intermediate in the dolichol-linked pathway to glycoproteins.