A fermentative hydrogen-producing bacterium was isolated from a food waste treatment process. The biological hydrogen production rate by the pure isolate (designated as Clostridium tyrobutyricum JM1) was dependent on various nutritional and environmental conditions. In this study, to enhance hydrogen production rate, the individual and mutual effects of three key process variables such as glucose concentration, pH and temperature were investigated through response surface methodology (RSM) in a batch system. A Box-Behnken design was employed to determine the effect of the three independent variables on the hydrogen production rate and to find the optimum condition of each variable for improved hydrogen production. Experimental results showed that a maximum hydrogen production rate of 5089 ml H-2 (g dry cell h) (1) was obtained under the condition of glucose concentration of 102.08 mM, temperature 35 degrees C and pH 6.5, and all three factors had significant influences on the specific hydrogen production rate. The RSM with the Box-Behnken design was a useful tool for achieving the high rate of hydrogen production by C. tyrobutyricum JM1. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.