We report the preparation of Langmuir-Blodgett (LB) films composed of oxidized carbon nanotubes (CNTs) and hydrogenase (H(2)ase) conjugates and their electrochemical properties. Both single-walled (SWNTs) and multi-walled CNTs (MWNTs) were used to form mixed monolayers with H2ase on the Tris-HCl subphase surfaces. By using the LB method, the CNTs-H(2)ase monolayers were transferred onto CaF2 and indium tin oxide (ITO) electrode surfaces. The LB film modified electrodes showed a couple of waves centered at around -500 mV (versus Ag/AgCl), which corresponding to the redox reaction of [4Fe-4S](2+/1+) clusters in the H(2)ase. The current intensity was enhanced after co-assembly with CNTs. Because of the different diameters of CNTs, this current intensity was proportional to the scan rate (v) for the electrodes modified with the LB films of pure H(2)ase and SWNTs-H(2)ase, but to the root of scan rate (v(1/2)) for those modified with the MWNTs-H(2)ase LB film. The products of diffusion coefficient and concentration ((DC)-C-1/2) increased in the order of pure H(2)ase, SWNTs-H(2)ase, and MWNTs-H(2)ase LB films. (c) 2006 Elsevier Ltd. All rights reserved.