A method based on isotope dilution-mass spectrometry was developed for the determination of nine cholesterol oxidation products in human plasma. The cholesterol oxidation products determined were cholest-5-ene-3 (\beta, 7 \alpha)-diol, cholest-5-ene- (3 \beta, 7 \beta)-diol ( (7 \alpha) - and (7 \beta)-hydroxycholesterol, respectively), (3 \beta)-hydroxycholest-5-en-7-one (7-oxocholesterol), 5,6 (\alpha)-epoxy(5 \alpha)-cholestan-3 (\beta)-ol (cholesterol-5 (\alpha, 6 \alpha)-epoxide), (5,6 \beta-) epoxy-5 (\beta)-cholestan-3 (\beta)-ol (cholesterol-5 (\beta, 6 \beta)-epoxide), cholestane-3 (\beta, 5 \alpha, 6 \beta)-triol, cholest-5-ene-3 (\beta, 24)-diol (24-hydroxycholesterol), cholest-5-ene-3 (\beta, 25)-diol (25-hydroxycholesterol), and cholest-5-ene-3 (\beta, 27)-diol (27-hydroxycholesterol). A corresponding deuteriumlabeled internal standard, containing 3 to 6 deuterium atoms, was synthesized for each cholesterol oxidation product except (5 \beta, 6 \beta)-epoxycholesterol which was determined using the internal standard for (5 \alpha, 6 \alpha) epoxycholesterol. Plasma from 31 healthy volunteers was analyzed by the new method and (27-, 24-), and (7 \alpha) hydroxycholesterol were the most abundant cholesterol oxidation products (mean values 154,64 , and (43 \mathrm{ng} / \mathrm{ml}), respectively). The other oxysterols determined were present in concentrations lower than (30 \mathrm{ng} / \mathrm{ml}). Males had higher 27-hydroxycholesterol concentrations in plasma than females. The 5,6-oxygenated products were present mainly unesterified while the other oxidation products were mostly in esterified form. 1995 Academic Press, Inc.