The oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7beta-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11beta-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7beta-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7beta-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7beta-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in humans.