Abstract The interaction of the flavoprotein glutathione reductase from yeast with NADH has been studied. A reduced enzyme species resulting from addition of 2 electrons per enzyme-bound FAD is formed on addition of NADH to oxidized enzyme. This 2-electron reduced enzyme is stable toward further reduction to the 4-electron reduced state by excess NADH. Yeast glutathione reductase reduced by excess NADH has spectral properties distinct from those of yeast glutathione reductase reduced by excess NADPH, the physiological substrate, but virtually identical with the spectral properties reported by Massey and Williams (J. Biol. Chem., 240, 4470 (1965)) for yeast glutathione reductase reduced by a 526-fold molar excess of GSH. It is concluded that glutathione reductase reduced by a small molar excess of NADH is the free, 2-electron reduced enzyme with no oxidized or reduced pyridine nucleotide bound to it. A bimolecular rate constant of 1.8 x 104 m-1 sec-1 was measured using stopped flow techniques for reduction of the enzyme-bound FAD by NADH. The rate of reduction of the enzyme-bound FAD by NADH is identical with the steady state rate of NADH-dependent GSSG reduction, suggesting that reduction of enzyme-bound FAD is rate limiting in the over-all reaction.