Production of Bio-Hydrogen in genetically modified bacteria or algae is an important future aim of industrial Biotechnology, but this currently is limited by the availability of oxygen stable hydrogenases. We report here the identification of a Citrobacter strain with strong hydrogen production under aerobic conditions. We have purified and identified the corresponding type II Ni/Fe hydrogenase and showed its in vitro Hydrogenase activity under normal atmosphere. We generated recombinant E. coli strains expressing the Hydrogenase operon and showed that these strains also produce hydrogen when cultivated in aerobic conditions. The identified enzyme may be useful for biological hydrogen production