To achieve sustainable hydrogen production by microbial electrolysis cell (MEC) without precious metal catalysts, we examined the potential of thermophilic microorganisms as biocatalysts on the cathode of MEC. A biocathode was firstly developed in a single-chambered MEC operated at 55 degrees C and further analyzed in a two-chambered MEC. Linear sweep voltammetry showed that the biocathode had a reducing activity significantly higher than the control electrodes (bioanode or non-inoculated electrode). At the potential of -0.8 V vs. SHE, the thermophilic biocathode produced a current density of 1.28 +/- 0.15 A m(-2) and an H-2 production rate of 376.5 +/- 73.42 mmol day(-1) m(-2), which were around 10 times higher than those of the non-inoculated electrode, with the cathodic H-2 recovery of ca. 70%. The molecular-phylogenetic analysis of the bacteria on the biocathode indicated that the community was comprised of six phyla, in which Firmicutes was the most populated phylum (77% of the clones in the 16S rRNA library). Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.