Glutathione reductase (GR, NADPH: oxidized glutathione oxidoreductase, EC 18.104.22.168) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as reducing cofactor. The aim of the present work was to purify and characterize GR from bovine liver. GR was purified using 2', 5' ADP-Sepharose 4B and DEAE-Sepharose Fast Flow columns. The enzyme has been purified 5456-fold and with a yield of 38.4%. The molecular and catalytic properties of bovine liver GR have been studied. Optimum temperature and pH was found to be 50 degrees C and 7, respectively. The activation energy of the reaction catalyzed by the enzyme was 9.065 kcal/mole. The molecular weight of the enzyme was found to be 55 kDa by SDS-PAGE. Kinetic characterization of bovine liver GR was also investigated, Km(NADPH) 0.063 +/- 0.008 mM and Km(GSSG) 0.154 +/- 0.015 mM were determined. It is accepted that parallel lines observed in these double reciprocal plots obeys Ping Pong mechanism and we have showed this in our steady state study. According to our results of statistical analysis, the Ping Pong mechanism is a suitable model since the loss function is less than the other mechanisms. However, competitive inhibition by a product could be accepted in sequential mechanisms but not in a Ping Pong mechanism. In this study, kinetic data are consistent with a branching reaction mechanism previously proposed for GR from other sources by other studies.