The electrochemical behavior of toluidine blue in solution has been studied and found to be strongly dependent on the electrode surface nature. Monolayers of cystamine (C) or 3-mercaptopropionic acid (MP) chemisorbed onto gold electrode surfaces have been used as support for covalent attachment of toluidine blue (TOB) through its aromatic amino group in position 3 and the amino or carboxylic groups covering the electrode surface. After covalent attachment of TOB onto an MP monolayer, three different species remained stably attached to the electrode surface. Physically adsorbed TOB, which showed the same midpoint potential as in the solubilized state, El,z = -24 j mV (Ag/AgCl); covalently attached TOB, whose midpoint potential was shifted toward 120 mV more positive potentials; and an unknown species, probably physically adsorbed TOB dimers, with a midpoinc potential, = +135 mV (Ag/AgCl). Toluidine blue modified electrodes started to oxidize catalytically NADH at -100 mV with an amperometric response dependent on the concentration of the substrate. An electrode fouling process was observed after successive additions of NADH leading to the loss of catalytic activity.