The present study investigated the effect of various dietary riboflavin supplementations (0 to 4000 mg/kg) during lactation on riboflavin concentrations of liver, carcass (bled body without intestine and liver), and milk in the rat. The experiment was conducted until the 14th day of lactation; milk samples were drawn on the 7th and 13th day of lactation. Riboflavin concentrations of milk raised continuously with increasing riboflavin supplementation; in the range between 0 and 10 mg/kg riboflavin supplementation, there was a linear relationship, and in the range between 12 and 4000 mg/kg there was a logarithmic relationship between riboflavin supplementation and riboflavin concentration in the milk. Maximum riboflavin concentration of milk obtained by supplementation with 4000 mg/kg was twelve-fold higher than without riboflavin supplementation. For riboflavin supplementation up to 12 mg/kg, riboflavin concentrations in milk on the 7th day of lactation and that on the 13th day of lactation were not different. In contrast, in rats fed diets with higher riboflavin supplementation, riboflavin concentrations were higher by 25% in average in milk on the 13th day of lactation than in milk on the 7th day of lactation. Contrary to the milk, riboflavin concentrations in liver and carcass exhibited a saturation, which was achieved at a supplementation of 6 mg/kg (liver) and 10 mg/kg (carcass), respectively. Maximum riboflavin concentrations obtained at a supplementation of 4000 mg/ kg were 1.9- and 2.3-fold higher for liver and carcass, respectively, than concentrations obtained without riboflavin supplementation. The dose-response relationship using riboflavin concentrations of liver and carcass as response factors indicates a riboflavin requirement of 8 to 9 mg/kg for lactating rats fed a semisynthetic diet with 17.4 MJ ME/kg dry matter and 20.8% protein in dry matter.