In this research, batch and continuous-flow reactors were used to study biological hydrogen production by anaerobic mixed communities subjected to two types of heat treatment--initial and repeated heat treatment. The bioreactors were inoculated with anaerobically digested municipal sludge, and sucrose was used as substrate. In the batch experiments, applying heat treatment at 1000C for 15 min. to the seed inoculum (initial heat treatment) was found necessary to sustain hydrogen production, and continuously applying heat treatment at 1000C for 15 min. to the sludge (repeated heat treatment) could promote higher hydrogen production. In the continuous-flow experiments, besides applying initial heat treatment at 1000C for 15 min. to the inoculum, repeated heat treatment at 900C for 20 min. was applied to the return sludge using external heated chamber. Results indicated that both initial and repeated heat treatments promoted hydrogen production by eliminating non-spore-forming, hydrogen-consuming microorganisms and by selecting for hydrogen-producing, spore-forming bacteria. It was also observed that the hydrogen production efficiency was superior at a higher feed concentration (0.13 L-H2/g COD at 20 g COD/L) in comparison to a lower feed concentration (0.07 L-H2/g COD at 6 g COD/L). Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that Clostridium and Bacillus species were dominant populations in the bioreactors. A positive correlation was observed between the total abundance of Clostridium species and hydrogen production during part of an operational run.