Hydrogenases are of great interest due to their potential use in H-2-based technology. However, most hydrogenases are highly sensitive to O-2, which have been the major bottleneck in hydrogenase studies. Here we report an O-2-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5 kDa. The enzyme showed a specific activity for H-2-oxidation of 661 U/mg, which is 35-fold greater than that for H-2-production of 18.7 U/mg. Notably, the MBH showed a remarkable O-2-stability, maintaining almost 95% of its original activity even after incubation for 30 h in air at 4 degrees C. These results suggest that the O-2-stable MBH may play an important role in the H-2-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O-2-stable MBH from the genus of Citrobacter. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.