Biohydrogen could efficiently be produced in glucose-fed biofilm reactors filled with plastic carriers and operated at 70°C. Batch experiments were, in addition, conducted to enrich and cultivate glucose-fed extremethermophilic hydrogen producing microorganisms from a biohydrogen CSTR reactor fed with household solid waste. Kinetic analysis of the biohydrogen enrichment cultures show that substrate (glucose) likely inhibited hydrogen production when its concentration was higher than 1 g/L. Different start up strategies were applied for biohydrogen production in biofilm reactors operated at 70°C, and fed with synthetic medium with glucose as the only carbon and energy source. A biofilm reactor, started up with plastic carriers, that were previously inoculated with the enrichment cultures, resulted in higher hydrogen yield (2.21 mol H2/mol glucose consumed) but required longer start up time (1 month), while a biofilm reactor direcdy inoculated with the enrichment cultures reached stable state much faster (8 days) but with very low hydrogen yield (0.69 mol H2/mol glucose consumed). These results indicate that hydraulic pressure is necessary for successful immobilization of bacteria on carriers, while there is the risk of washing out specific high yielding bacteria. © 2008 Wiley Periodicals, Inc.