The authors have developed a fluorescence assay to measure the rate and extent of sterol oxidation in lipid bilayers. Dehydroergosterol (DHE), a fluorescent cholesterol analog, is used as a probe and at the same time as a membrane component. The assay can also be performed on bilayers containing a mixture of sterols including DHE and nonfluorescent sterols, such as cholesterol and ergosterol. The fluorescence intensity of DHE decreases on oxidation, so the rate and extent of free radical- or enzyme-induced sterol oxidation can be measured as a function of temperature and membrane composition. For the studies, two-component (e.g., phosphatidylcholine (PC)/DHE) and multicomponent (e.g., DHE/PC/bovine-brain sphingomyelin) large unilamellar vesicles were used, and sterol oxidation was initiated either by the peroxy radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride or by the enzyme cholesterol oxidase. The data gathered from this assay may be used to examine the effects of water- and lipid-soluble antioxidants on membrane sterol oxidation produced by free radicals. This assay can be used to test the potency of antioxidants and pro-oxidants, and can be used to determine whether unknown substances demonstrate antioxidant activity against sterol oxidation. The assay can also be used as a tool to examine the effect of sterol lateral organization on sterol oxidation (in the presence or absence of antioxidants). In agreement with the sterol regular distribution model, it is found that both free radical- and enzyme-induced sterol oxidation vary with membrane sterol content in a well defined alternating manner.