Microcalorimetry, spectrophotometry, and high-performance liquid chromatography (h.p.l.c.) have been used to conduct a thermodynamic investigation of the glutathione reductase catalyzed reaction {2 glutathione(red)(aq)+NADP(ox)(aq)=glutathione(ox)(aq)+NADP(red)(aq)}. The reaction involves the breaking of a disulfide bond and is of particular importance because of the role glutathione(red) plays in the repair of enzymes. The measured values of the apparent equilibrium constant K' for this reaction ranged from 0.5 to 69 and were measured over a range of temperature (288.15 K to 303.15 K), pH (6.58 to 8.68), and ionic strength I-m (0.091 mol . kg(-1) to 0.90 mol . kg(-1)). The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations led to values of thermodynamic quantities at T = 298.15 K and I-m = 0 for a chemical reference reaction that involves specific ionic forms. Thus, for the reaction {2 glutathione(red)(-) (aq) + NADP(ox)(3-)(aq) glutathione(ox)(2-)(aq) + NADP(red)(4-) (aq) + H+ (aq)}, the equilibrium constant K = (6.5 +/- 4.4) . 10(-11), the standard molar enthalpy of reaction Delta(r)H(m)(o) = (6.9 +/- 3.0) kJ . mol(-1), the standard molar Gibbs free energy change Delta(r)G(m)(o) = (58.1 +/- 1.7) kJ . mol(-1), and the standard molar entropy change Delta(r)S(m)(o) = -(172 +/- 12) J . K-1 . mol(-1). Under approximately physiological conditions (T = 311.15 K, pH = 7.0, and I-m = 0.25 mol . kg(-1) the apparent equilibrium constant K' approximate to 0.013. The results of the several studies of this reaction from the literature have also been examined and analyzed using the chemical equilibrium model. It was found that much of the literature is in agreement with the results of this study. Use of our results together with a value from the literature for the standard electromotive force E-o for the NADP redox reaction leads to E-o = 0.166 V (T = 298.15 K and I = 0) for the glutathione redox reaction (glutathione(ox)(2-) (aq) + 2 H+(aq) + 2 e(-) = 2 glutathione(red)(-)(aq)}. The thermodynamic results obtained in this study also permit the calculation of the standard apparent electromotive force E-'o for the biochemical redox reaction {glutathione(ox)(aq) + 2 e(-) = 2 glutathione(red)(aq)} over a wide range of temperature, pH, and ionic strength. At T = 298.15 K, I = 0.25 mol . kg(-1), and pH = 7.0, the calculated value of E-'o is -0.265 V. Published by Elsevier Science Ltd.