Increases in cytochrome P-450 and cytochrome b5 and a decrease in NADPH-cytochrome c (P-450) reductase were generally brought about by feeding the riboflavin-deficient diet to young rats (50-120 g body weight) for 5 weeks, whereas no significant changes in these enzymes were observed with rats of 220 g body weight by feeding for 2 weeks. Amounts of lipid peroxides in the serum or the liver tissue and microsomes increased significantly after feeding the deficient diet for 5 weeks, in comparison with the respective control rats. On the other hand, NADPH-dependent lipid peroxidation in the presence of ferric ion and pyrophosphate, assayed as malondialdehyde, was decreased drastically in the liver microsomes of all groups of riboflavin-deficient rats irrespective of the period or body weight of animals. Lipid peroxidation could be detected by addition of EDTA-ferric ion or ferricyanide to the incubation medium, though the formation of malondialdehyde was less than that expected by the activity of NADPH-cytochrome c reductase. NADPH-reduction of nitroblue tetrazolium with liver microsomes was decreased in riboflavin deficiency, and was almost able to be correlated with the activity of NADPH-cytochrome c reductase. Following intraperitoneal injection of riboflavin into the deficient rats, NADPH-dependent lipid peroxidation in the presence of ferric ion and pyrophosphate recovered only to 10% of the control rates at 40 hr after the injection, when cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase levels were restored to those of their respective controls. Activities of drug-metabolizing enzymes, aminopyrine demethylase and aniline hydroxylase were decreased by initiation of feeding from the weanling stage, but the activities changed only slightly by feeding from the 120 g of body weight stage.