The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H-2 (g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E aerogenes. (c) 2007 Elsevier Ltd. All rights reserved.