The placental transfer of cholesterol (5-cholesten-3beta-ol) was investigated by giving pregnant rhesus monkeys cholesterol-1alpha-(3)H or cholesterol-4-(14)C and then determining the cholesterol specific activity (SA) in maternal serum and in fetal serum and tissues. An isotopic steady state was established in five pregnant animals by the daily feeding of a tracer dose of cholesterol-4-(14)C. Comparison of maternal and fetal serum cholesterol SA revealed that an average of 42.6% of the serum cholesterol in the term fetus originated by transfer from the maternal blood. The remainder presumably arose by fetal synthesis de novo. Fetal tissues had cholesterol SA equal to or slightly less than that of fetal serum, except for brain which had a SA only 5% that of fetal serum. In other studies a single intravenous dose of radioactive cholesterol was given to either mother or fetus in late pregnancy. The time for detectable passage across the placenta in either direction was between 4 and 24 hr. With maternal administration of the isotope, there was equilibration of maternal and fetal serum cholesterol SA after 10-12 days. With fetal injection of isotopic cholesterol, however, the maternal cholesterol SA never attained a level more than 5% of fetal SA. This indicated that the net cholesterol flux was strongly in the direction of mother to fetus. Serum cholesterol levels were significantly greater in maternal than in fetal serum (80.3+/-18.5 vs. 59.6+/-15.6 mg/100 ml). Maternal serum cholesterol concentration in the monkey was significantly lower in late pregnancy than during the puerperium. Studies of breast milk indicated that approximately two-thirds of milk cholesterol was transferred from the maternal blood.