The activity of NADH oxidase from Thermus thermophilus (NADox) was examined by spectrophotometric techniques following the kinetics of NADH oxidation at 340 nm in the presence of the enzyme cofactor the Flavin adenine mononucleotide (FMN), the ferricinium cation (C(5)H(5))(2)Fe(+), the mixture of the two or in the absence of both. Contrary to previous results, the enzyme is active in the absence of FMN for the oxidation of NADH using its natural electron acceptor the oxygen. This activity is enhanced 40 times in the presence of FMN and more than 100 times in the presence of the ferricinium cation (FcH(+)). In the latter case, the addition of FMN produces no increase in the activity demonstrating the advantage of using FcR(+) as redox mediator for the oxidation of NADH in the presence of NADox. This advantage has been used to develop two disposable NADH biosensors based on NADox, one operating in anodic mode by combining the enzyme with ferrocene derivatives (FcR) and the other in cathodic mode when the enzyme is associated to ferricinium cations. The two biosensors display very good sensitivities and the alternative use of one or the other can cover an extensive linear range for NADH detection. (C) 2010 Elsevier B.V. All rights reserved.