Weanling male rats were fed a riboflavin- or selenium-deficient diet for 5 weeks, and the glutathione content and its relating enzyme activities in the livers were examined. The glutathione content and glutathione reductase activity were decreased by deficiency of riboflavin, but not by that of selenium. Glutathione peroxidase activity was increased by addition of selenium to the diet, but without its addition, the activity was higher in the riboflavin-deficient rats than in the riboflavin-sufficient rats, in spite of the increase of lipid peroxides in the former rats. Glucose 6-phosphate dehydrogenase activity was decreased significantly by riboflavin deficiency. In another experiment, riboflavin was intraperitoneally injected into rats fed the diet to which neither riboflavin nor selenium had been added. The glutathione content and activities of glutathione reductase and glucose 6-phosphate dehydrogenase returned to the control level of riboflavin-sufficient rats in 24 h, the lipid peroxide level in 48 h, and the glutathione peroxidase activity, being higher than that in the control rats, in 72 h after the injection, respectively. These findings indicate that the increase of lipid peroxides in the livers of riboflavin-deficient rats is caused by the decrease in the glutathione content as well as glutathione reductase activity rather than by that in the selenium-dependent glutathione peroxidase activity.