Purified cholesterol is quite unstable when stored in air at room temperature. Products of cholesterol auto-oxidation were concentrated from several lots of USP-grade cholesterol by recrystallizing cholesterol from the methanol extract, retaining the mother liquor, and evaporating the residuum to dryness under vacuum. By application of TLC and GLC, major, individual, oxidation compounds were identified and quantitated. Biological activities of these oxidation compounds were studied by using cultured rabbits'' aortic smooth muscle cells. The concentrate of the auto-oxidation products of cholesterol showed remarkable in vitro cytotoxic effects, whereas purified cholesterol at the same concentration produced no toxic effects. The concentrate was further separated into 6 TLC fractions. 25-Hydroxycholesterol and cholestane-3.beta., 5.alpha., 6.beta.-triol were probably responsible for the biological toxicity of the concentrate.