Hydrogenase (H(2)ase)-cationic electrolyte biohybrids were assembled at the air-water interface via intermolecular electrostatic interaction. The H(2)ase used was purified from the phototropic bacterium of Thiocapsa roseopersicina. Two kinds of cationic electrolyte compounds (CECs) were used, the difference of which was whether they contained viologen substituent or not. Surface pressure-area isotherms indicated that these CECs were co-existed with the H(2)ase in the monolayers, which were then transferred to substrate surfaces to form H(2)ase-CECs hybrid films by the Langmuir-Blodgett (LB) method. Uniform film was formed when polyelectrolyte was used as the subphase. Cyclic voltammograms (CVs) of the LB films showed a couple of redox waves in the potential range of -0.4 to -0.65 V vs. Ag/AgCl, which was ascribed to one electron process of either [4Fe-4S] clusters of H(2)ase or viologens of the CECs. A direct electron transfer between the H(2)ase and electrode surface was achieved in the LB films. Stronger current intensity was recorded when the CV measurements were done in H-2 saturated electrolyte solution than that in Ar. It was confirmed that the H(2)ase biocatalytic activity remained in the LB films. Thus, we suggest that the present H(2)ase-CECs biohybrids could act as potential materials for the studies of interconversion reaction of H-2 and protons. (C) 2010 Elsevier B.V. All rights reserved.