When LDL is oxidized by a free radical generating system, both cholesterol and fatty acyl moieties within the LDL are modified. Oxidized low density lipoprotein (oxLDL) can induce alterations in the Ca2+ transients in isolated cardiomyocytes. It is unclear if oxidation of the LDL fatty acyl chains (in phospholipids, triglycerides and cholesteryl esters) or oxidation of the LDL unesterified cholesterol is more important in producing the oxLDL-induced alteration in cellular calcium transients. Therefore, we investigated the possible role of oxidized cholesterol and fatty acyl chain peroxidation in the effects of LDL on Ca2+ transients. Cholesterol oxidase (CO) treatment of LDL produced oxidized cholesterol plus H2O2. The H2O2 peroxidized the LDL fatty acyl chains, as indicated by an increased malondialdehyde (MDA) content. The cell systolic [Ca 2+] was significantly increased after incubation with CO-treated LDL. Diastolic [Ca2+] was unchanged. MDA content in the CO-treated LDL correlated with the change in systolic [Ca2+] of treated cells. Catalase, a scavenger of H2O2, inhibited MDA formation in the CO-treated LDL and prevented the increment in systolic [Ca2+] in the treated cells. A similar stimulatory effect on the Ca2+ transient was observed if cells were treated with LDL after exposure to only H 2O2 and not CO. Direct exposure of myocytes to H 2O2 (without LDL) failed to produce a stimulatory effect on the calcium transient but caused an increment in cellular diastolic Ca 2+. Exposure of myocytes to CO alone (without LDL) produced a significant increment in diastolic [Ca2+] and this effect was not prevented by catalase. These results suggest that fatty acid peroxidation in the LDL moiety is more important than oxidized cholesterol in the generation of ox-LDL-induced increases in Ca2+ transients in isolated cardiomyocytes. Further, oxidation of in situ cell membrane cholesterol will destroy cell integrity. Our data also underline the importance of adding extracellular lipid in any study of the effects of oxygen free radicals on cellular function. Under conditions of radiologic or chemically-induced generation of oxygen-derived free radicals, cardiac dysfunction and damage may be induced by this process.