Aims: To profile the fractions of bacteria in heat- treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. Methods and Results: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene- directed polymerase chain reaction denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis ( 99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H-2 g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. Conclusion: Clostridium species in heat- treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. Significance and Impact of the Study: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.