Female rats at various stages of pregnancy were injected intraperitoneally with [3H]water; 4 h later, they were killed, the uterus was removed, and the fetuses were dissected. Lipids were isolated and fractionated by HPLC and the total amount of cholesterol in each organ, as well as radioactivity incorporated into cholesterol and cholesterol precursors, were determined. From the data for cholesterol content at each age we calculated the rate of accumulation of cholesterol during fetal development. As incorporation of label from [3H]water takes place with a stoichiometry defined by a known biosynthetic pathway, we were also able to determine the fraction of cholesterol accumulating in each organ that had been newly synthesized. For the fetus as a whole, more than 93% of the cholesterol accumulating during development was newly synthesized. As the specific radioactivity of cholesterol in the maternal circulation was negligible (because synthesis of cholesterol by maternal liver was suppressed by inclusion of cholesterol in the diet), we conclude that the fetus synthesizes nearly all of its own cholesterol; neither the maternal circulation nor the placenta/yolk sac contribute significant amounts of cholesterol to the fetus. We were also able to quantitate trafficking of cholesterol between fetal organs. Fetal brain is responsible for the synthesis of all of its own cholesterol. In contrast, fetal liver exports cholesterol into the fetal circulation and supplies about half of the cholesterol for development of heart, lung, and kidney.