Biohydrogen production by anaerobic microbes enriched from heat-shocked cow dung compost was studied by using an artificial medium containing sucrose. Initial pH and substrate levels were selected as target factors in this study. Our experimental results demonstrated that optimal substrate concentration and pH for the composts generating hydrogen gas were 4.0 +/- 0.5 g sucrose/l and 5.4 +/- 0.2, respectively. Supplementary experiments confirmed that chemical oxygen demand reduction efficiency (69%) obtained from the conditions of sucrose = 4.0 g/l and pH = 5.5 was significantly greater than that (37%) from sucrose = 5.0 g/l and pH = 5.0. Experimental results of metabolites analysis led us to the conclusion that Clostridium sp. predominated in the anaerobic composts, suggesting that inocula used to seed the batch experiment can be obtained from a common natural source. (C) 2003 Elsevier Ltd. All rights reserved.