The glassy carbon electrode modified with nafion and toluidine blue O (TBO) was prepared and their electrochemical properties were investigated. The TBO molecules were strongly and irreversibly adsorbed on the nafion-modified glassy carbon surface. The electrochemical behavior and mechanism for interactions of the TBO molecules with the nafion film were investigated through cyclic voltammetric and U-V-vis spectra methods. The electrocatalytic reductions of nitric oxide and oxygen were performed at this modified electrode by cyclic voltammetry, rotating ring disk voltammetry and chronoamperometry as diagnostic techniques. The nafion membrane played a duel role as a matrix for the TBO immobilization and also helped to partitioning the NO from the solution phase. The diffusion coefficients of nitric oxide and oxygen at the TBO/nafion/GC modified electrode were calculated using chronoamperometric technique and rotating ring disk voltammetry, respectively. The W value for nitric oxide and oxygen obtained by the above methods were found as 1.2798 x 10(-6) and 1.4 x 10(-5) cm(2) s(-1), respectively. From the intercept value of the Koutecky-Levich plot, the catalytic reaction rate constant, k, was calculated for O-2 reduction and found as 6.6 x 10(4) M-1 S-1. (c) 2006 Elsevier B.V. All rights reserved.