These studies used the suckling lamb as a model to determine the sources of cholesterol that are utilized for development of the central nervous system in the neonate. Lambs were studied at 1.3 and 16.4 days after birth. Over this 15-day interval, 14 g of new brain tissue were formed. About 9-10 mg of cholesterol were utilized daily for this new tissue growth. To determine the source of this cholesterol, the absolute rates of low-density lipoprotein clearance and cholesterol synthesis were measured in vivo in nine separate regions of the central nervous system. Low-density lipoprotein clearance throughout the brain was very low and at most could have contributed only 0.3-0.4 mg cholesterol daily for new brain growth. In contrast, the brain synthesized 7-8 mg of cholesterol/day. There were pronounced regional differences in the concentration of cholesterol throughout the brain, and these correlated closely with the rate of sterol synthesis (r = 0.95) in these same regions. We conclude that the principal source of sterol for brain growth in suckling lambs is de novo synthesis.