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 β,7 α-diol, cholest-5-ene-3 β,7β-diol (7α- and 7β-hydroxycholesterol, respectively), 3β-hydroxycholest-5-en-7-one (7-oxocholesterol), 5,6α-epoxy-5α-cholestan-3β-ol (cholesterol-5α,6α-epoxide), 5,6β-epoxy- 5β-cholestan-3β-ol (cholesterol-5β,6β-epoxide), cholestane-3β,5α,6β-triol, cholest-5-ene-3β,24-diol (24-hydroxycholesterol), cholest-5-ene-3β,25-diol (25-hydroxycholesterol), and cholest-5-ene-3β,27-diol (27-hydroxycholesterol). A corresponding deuterium-labeled internal standard, containing 3 to 6 deuterium atoms, was synthesized for each cholesterol oxidation product except 5β,6β-epoxycholesterol which was determined using the internal standard for 5α,6α-epoxycholesterol. Plasma from 31 healthy volunteers was analyzed by the new method and 27-, 24-, and 7α-hydroxycholesterol were the most abundant cholesterol oxidation products (mean values 154, 64, and 43 ng/ml, respectively). The other oxysterols determined were present in concentrations lower than 30 ng/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.