Bioelectrocatalytic hydrogen (H-2) production was studied using Thiocapsa roseopersicina hydrogenase in a two-compartment proton-exchange-membrane (PEM) fuel-cell system equipped with carbon-paper electrodes. Sodium dithionite (SID), as an electron donor, and hydrogenase, as a catalyst, were used in the anodic oxidation reaction and in the cathodic reduction reaction, respectively. Methyl viologen (MV) was added for the electron relays in both reactions, The concentrations of phosphate buffer, MV and hydrogenase in the reaction chambers were optimized, in which the concentration of SD was fixed at 20 mM. Parameters including the cathode surface area, the distance between electrodes, and the external load were optimized to complete the system. Catalytic current generation in the cathode increased from 0.12 to 0.19 mA and from 0.07 to 0.12 mA, in proportion to the hydrogenase concentration (34.8-347.5 mu g/mL) and the cathode surface area (2.0-11.5 cm(2)), respectively; however, it decreased from 0.37 to 0.08 mA and from 0.12 to 0.09 mA with the increase of the electrical load (5-1000 Omega) and the distance between electrodes (1.5-3.5 cm), respectively. The optimal MV concentrations were 2.5-5 mM in the cathode chamber. The bioelectrocatalytic H, production rate was calculated from the cathodic current in argon atmosphere, and the maximal value under the partially optimized conditions was estimated to be 0.16 mu mol H-2/min/mg-protein, which was less than 8% of the specific H-2 production activity, 2.20 mu mol H-2/min/mg-protein, of the hydrogenase purified from the cytoplasmic fraction of T. roseopersicina. This study indicates that T. roseopersicina hydrogenase has a high potential for bioelectrocatalytic H-2 production; still, much effort could be required to develop a proper biofuel-cell system that provides for efficient transfer of elections and protons. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.