Increases or decreases of red cell glutathione reductase (GR) have been described in connection with many clinical abnormalities. We find that GR activity as measured in hemolysates represents only a portion of the available GR activity. The addition of small amounts of flavin adenine dinucleotide (FAD), but not of flavin mononucleotide or riboflavin, activates the GR of hemolysates. 1 μM FAD results in a maximal activation within 10 min; gradually increasing activation occurs at much lower, for example, 20 mμM FAD concentrations. Once FAD has activated GR, dilution or dialysis does not reverse activation of the enzyme. Activation of GR by FAD can be inhibited by adenosine triphosphate (ATP), and to a lesser extent by adenosine diphosphate (ADP) and adenosine monophosphate (AMP), if these adenine nucleotides are added before the addition of FAD, but only to a slight extent if FAD is added before the adenine nucleotides. The addition of FAD to GR does not alter its electrophoretic mobility but produces intensification of the bands.