The regeneration of NADH in a batch electrochemical reactor using a ruthenium modified glassy carbon electrode (RuGC) has been investigated. The information on the structure of the electrode/electrolyte interface in the presence of NAD(+) in the solution, the kinetics of NAD(+) reduction, and the batch-electrolysis NADH regeneration has been obtained using electrochemical techniques of dc linear potential (LP) and constant potential (CA) polarization, ac differential capacitance (DC), and electrochemical impedance spectroscopy (EIS). It has been shown that the modification of GC by a sub-monolayer of Ru can provide an electrode surface capable of reducing NADI directly to NADH at a high yield of enzymatically active 1,4-NADH (96%). From the electrochemical point of view, the reaction is irreversible and occurs at high cathodic overpotentials, where the reaction rate is controlled by the surface diffusion of electroactive species. EIS measurements have shown that the electrode/electrolyte interface and the corresponding charge- and mass-transfer processes can be described by an electrical equivalent circuit composed of two time constants in parallel, with the additional contribution of a mass-transport Warburg impedance element. The time constant recorded at higher frequencies represents the response of a GC part of the electrode surface, while the lower-frequency time constant can be related to the response of Ru sites on the electrode surface. It has been determined that the NAD+ reduction reaction is of first order with respect to NADI. The calculated apparent heterogeneous reaction rate constant values are rather low, which is due to the slow mass-transport of electroactive species at the electrode surface. The kinetic analysis has demonstrated that a very good agreement between the apparent heterogeneous reaction rate constant values calculated using three different experimental techniques is obtained. (C) 2004 Elsevier B.V. All rights reserved.