Male Wistar rats received intraperitoneal injections of adriamycin (4 mg/kg body weight/day) and/or riboflavin-butyrate (20 mg/kg body weight/day) for 6 consecutive days. Cardiac mitochondria were then prepared for our present experiment. The combined use of riboflavin-butyrate with adriamycin was evaluated for reduction of lipid peroxide formation in rat cardiac mitochondria. In order to find the mechanism of the effect of riboflavin-butyrate, the glutathione peroxidase-glutathione reductase system was examined. Adriamycin reduced the glutathione reductase activity in rat cardiac mitochondria, but did not affect the glutathione peroxidase activity. The mitochondrial content of flavin adenine dinucleotide, a prosthetic group of glutathione reductase, was greatly reduced and apoprotein of glutathione reductase also decreased. The administration of riboflavin-butyrate with adriamycin increased flavin adenine dinucleotide and glutathione reductase activity. These results suggest that exogenous administration of riboflavin-butyrate is capable of reducing lipid peroxide by both enzymatic detoxification through glutathione reductase and non-enzymatic detoxification due to direct reaction with lipid peroxide.