Hydrogenases are the key enzymes for hydrogen metabolism in many microorganisms. Due to the high efficiency they develop for H-2 oxidation, research in the last five years has aimed towards their use as biocatalysts for H-2/O-2 biofuel cells to replace platinum-based chemical catalysts. We report in this review the major issues that have been addressed in view of the future development of such a novel biotechnological device. This includes enhancing the stability of either the enzyme itself or its immobilization onto conductive supports, increasing the amount of electrically connected enzymes and, finally, controlling hydrogenase orientation at the electrode surface, and hence the electron transfer process. We specifically focus on a particular [NiFe] membrane-bound hydrogenase purified from the hyperthermophilic and microaerophilic bacterium Aquilex aeolicus. This enzyme resists to O-2, CO, and high temperatures making it potentially efficient as a biocatalyst. Recent progress in these domains strengthens the credibility of a viable H-2/O-2 biofuel cell and opens new avenues for biofuel cell design. (C) 2011 Elsevier Ltd. All rights reserved.