Important issues in the microbiology of biological reactors include bacterial composition and cell counts of bacterial communities. Because oligonucleotide probes targeting a particular phyletic group are available for studying hydrogen fermentation, fluorescence in situ hybridization (FISH) could be an attractive research technique. Although we reported the importance of the Megasphaera spp. for a simple hydrogen fermentation system, fundamental information like population and composition remains unclear. Thus, here, we established the use of Megasphaera-specific oligonucleotide Mega-X for FISH and the optimum conditions for rapid single-cell level identification. The culture isolated from a simple hydrogen fermentation process was identified as Megasphaera elsdenii and was shown to be significantly dominant (30%-58% of the microflora) by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and FISH. The combination of Megasphaera-specific PCR-RFLP and FISH is likely to become an important methodology for understanding the global distribution and behaviour of Megasphaera spp. in the environment. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.