Human lens membranes contain the highest cholesterol content of any known biological membrane. Although cholesterol is prone to oxidation, the presence of its oxidation products in human cataract has not been shown before. This study was designed to investigate the presence of cholesterol oxides in human cataractous lenses. Human clear lenses (n = 48) were obtained from Coimbra University Hospital Eye Bank. Human cataracts (n = 54) were obtained by routine extracapsular surgery. Cholesterol oxides were isolated by solid-phase extraction on a C18 cartridge and quantified as TMS-ether derivatives by gas chromatography. The extraction procedure allows for an efficient recovery of the major cholesterol oxides, while retaining virtually all cholesterol. Exposure of membranes isolated from transparent human lenses to the free radical generator 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) produced 7 alpha-hydroxycholesterol (6%), 7 beta-hydroxycholesterol (19%), 5 alpha, 6 alpha-epoxycholestanol (1%) and 7-ketocholesterol (74%) as major oxidation products. Cataractous lenses contained quantifiable amounts of 7 beta-hydroxycholesterol (7.3 +/- 0.74 mmol mol-1 cholesterol), 7-ketocholesterol (4.2 +/- 0.32 mmol mol-1 cholesterol), 5 alpha, 6 alpha-epoxycholestanol (0.9 +/- 0.16 mmol mol-1 cholesterol), 20 alpha-hydroxycholesterol (0.6 +/- 0.13 mmol mol-1 cholesterol) and 25-hydroxycholesterol (0.1 +/- 0.02 mmol mol-1 cholesterol), whereas clear lenses contained no detectable amounts of cholesterol oxides. We have shown, for the first time, that oxysterols accumulate in human cataracts. Although the total amount of oxidized cholesterol in cataracts is not likely to be high it may account for much of the membrane damage associated with cataract formation.