Isolated human low density lipoprotein (LDL) was oxidized with either cupric ions or soybean lipoxygenase and linoleic acid. Cholesterol oxidation products (oxysterols) were determined by isotope dilution gas chromatography-mass spectrometry. A new cholestane-3,5,6-triol isomer, cholestane-3 beta,5 alpha,6 alpha-triol, which has not previously been recognized as a cholesterol autoxidation product, was found at similar concentrations as the well-known cytotoxic cholestane-3 beta,5 alpha,6 beta-triol during both copper- and lipoxygenase-mediated LDL oxidation. Furthermore, two epimeric cholest-5-ene-3 beta,4-diols were identified in the oxidized LDL at similar concentrations. These two isomers were also identified in human atherosclerotic tissue in a ratio of 1:1 at a concentration more than 10-times higher than in non-atherosclerotic vessels. In vitro oxidation of LDL under an 18O2 atmosphere revealed that molecular oxygen was the only source of the oxygen functions at C-4 in the cholest-5-ene-3 beta,4-diols. Taken together, these findings suggest that the cholest-5-ene-3 beta,4-diols in atherosclerotic plaques are formed by autoxidation.