The major oxysterols in human circulation are 7 alpha-, 27-, and (24S)-hydroxycholesterol. Two unique experiments were performed to elucidate their origin and kinetics. A volunteer was exposed to (18)O(2)-enriched air. A rapid incorporation of (18)O in both 7 alpha- and 27-hydroxycholesterol and disappearance of label after exposure were observed. The half-life was estimated to be less than 1 h. Incorporation of (18)O in (24S)-hydroxycholesterol was not significant. In the second experiment a volunteer was infused with liposomes containing 10 g of [(2)H(6)]cholesterol. This resulted in an enrichment of plasma cholesterol with (2)H of up to 13%, and less than 0.5% in cerebrospinal fluid cholesterol. The content of (2)H in circulating 7 alpha-hydroxycholesterol remained approximately equal to that of plasma cholesterol and decreased with a half-life of about 13 days. The (2)H content of circulating 27-hydroxycholesterol was initially lower than that of cholesterol but in the last phase of the experiment it exceeded that of cholesterol. No significant incorporation of (2)H in (24S)-hydroxycholesterol was observed. It is evident that 7 alpha-hydroxycholesterol must originate from a rapidly miscible pool, about 80% of 27-hydroxycholesterol from a more slowly exchangeable pool, and more than 90% of (24S)-hydroxycholesterol from a nonexchangeable pool, presumably the brain. The results are discussed in relation to the role of oxysterols in cholesterol homeostasis and their use as markers for pathological conditions. - Meaney, S., M. Hassan, A. Sakinis, D. Lütjohann, K. von Bergmann, A. Wennmalm, U. Diczfalusy, and I. Björkhem. Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study. J. Lipid Res. 2001. 42: 70;-78.