Hydrogen (H-2) production by mutants of Enterobacter aerogenes HU-101, a strain isolated and characterized in our laboratory, was found to be enhanced compared with that of HU-101 due to blockage of production of other metabolites. The mutants were isolated by the allyl alcohol (AA) and/or proton suicide method. Among the AA resistant mutants isolated after NTG treatment of E. aerogenes HU-101, strain A-1 produced 78 mmol of H-2/l medium together with no 2,3-butanediol and reduced ethanol formation, compared to 52.5 mmol H-2/l medium by HU-101 when a complex medium containing 20 g/l glucose, 5 g/l yeast extract and 5 g/l tryptone was used for the cultivation, However, the amounts of lactate and acetate produced by A-1 were higher than those of HU-101. Subsequently, mutants incapable of producing these acids were isolated by a proton suicide method. A mutant (HZ-3) derived from HU-101 showed reduced acid production and increased H-2 production, to 1.5-fold higher than that of HU-101. Furthermore, the production of acidic and alcoholic metabolites by the mutant AY-2, obtained by treatment of A-1 with the proton suicide method was found to be lower, and its production of H-2 at 101.5 mmol/l medium, to be 2.0-fold higher, than those of HU-101.