In this work a thorough study of the oxidation of reduced glutathione (GSH) by electro-generated Berlin Green (BG) at Prussian blue (PB) film-modified glassy carbon electrode (GCE) was attempted by employing cyclic voltammetry (CV) and rotating-disc electrode (RDE) techniques. It has been shown that oxidation of GSH occurs at the potential coinciding with that of Fe-II(CN)(6) to Fe-III(CN)(6) transformation in the PB film, where no oxidation signal is observed at a bare GCE. The kinetics of catalytic reaction was investigated using a rotating-disc electrode voltammetry. The results obtained for various thicknesses of film and GSH concentrations are explained using the theory of electrocatalytic reactions at chemically modified electrodes (Andrieux-Saveant model) and it was concluded that the reaction has a "surface" reaction mechanism in which a few monolayers at film/solution side engaged in the catalytic process. However, the "surface" reaction tends to a saturation limit with increasing GSH concentration was observed and the behavior has been explained by using Michealis-Menten inner sphere kinetics. Tafel plots for various concentrations of GSH have been drawn and the slope values of 95-110 mV/decade indicate that the first electron transfer is not rate limiting process. The reaction order with respect to GSH and H+ were calculated as 0.6 and -0.4, respectively. (C) 2009 Elsevier Ltd. All rights reserved.