Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.