Recently, cholesterol hydroperoxides have been shown to be sensitive pathogenic markers of reactive oxygen species (ROS)-mediated damage though they have never been measured in heart tissue. We hypothesized that cholesterol hydroperoxides and oxysterols, putative cardiotoxic products of cholesterol oxidation, are elevated in the hearts of alcoholics as a consequence of ROS-mediated reactions. To test this, we measured 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH) by HPLC with postcolumn chemiluminescence as well as 7alpha- and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH) and 3beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto) by HPLC-UV in cardiac muscle of alcohol-fed rats. Alcohol feeding was carried out using a pair-feeding protocol with 35% of total dietary energy as ethanol; controls were pair-fed isocaloric glucose. After 6-7 wk treatment with alcohol, heart 7alpha-OOH, 7beta-OOH and 7beta-OH were significantly greater than in controls. Levels of heart phospholipid 16:0 and 18:1 were lower than in controls, while 18:0 and 18:2 were greater. This is the first report of the presence of 7alpha-OOH, 7beta-OOH and 7alpha-OH in cardiac tissue. The elevations in 7alpha-OOH and 7beta-OOH as well as 7beta-OH are evidence of increased oxidative stress and possible membrane changes. Alterations in the proportions of 16:0, 18:1, 18:2 and 18:0 in heart phospholipids provide further evidence of an altered membrane domain.