Rats treated chronically with the anticancer agent adriamycin exhibited lipid peroxides in the serum and hyperlipidemia. Independent assay methods based on thiobarbituric acid reactivity and iodometric titration confirmed the presence of both lipid endoperoxides and hydroperoxides. Fractionation studies indicated that lipid peroxides were mainly associated with neutral lipids, including triglycerides, cholesterol esters and cholesterol, rather than with phospholipids. The lipid peroxides were distributed throughout the major serum lipoprotein classes. Although the origin of the lipid peroxides has not been established, the dose dependence for elevation of serum lipid peroxides approximately corresponds to that required for the development of cardiomyopathy. These findings support the conclusion that lipid peroxides are formed in vivo in rats treated chronically with adriamycin.