A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive tricarboxylic acid cycle and grows rapidly with a generation time of about 1 h. TK-6 is believed to have an efficient hydrogen-oxidizing ability to support such rapid growth. We cloned hydrogenase genes from TK-6 and found that this strain has at least four clusters of hydrogenase genes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that all four hydrogenase gene clusters were transcribed under aerobic condition at hydrogen concentrations of 45% and 60%. One of them was not transcribed at a hydrogen concentration of 20%. All the four hydrogenase gene clusters were expressed under anaerobic denitrifying condition at a hydrogen concentration of 75%.