To measure the activation coefficient (AC) of erythrocyte glutathione reductase (GR), suspended whole blood was lysed in a preincubation solution, with or without 100 microM flavin adenine dinucleotide (FAD). Upon addition of the reaction mixture, FAD concentration decreased about 10-fold. No AC values < 1 were obtained in any of the subjects. The range of unstimulated activity per g hemoglobin (Hb) was 5 to 12 U. AC values of healthy subjects (1.3) decreased to about 1.15 after vitamin supplementation of 1 RDA for 3 wk. In healthy young subjects consumption of dietary riboflavin at levels as low as 0.5 mg/d resulted in an AC of 1.6.