Two regions of the Methanobacterium thermoautotrophicum genome containing genes that encode enzymes involved in methanogenesis (methane genes) have been cloned and sequenced to determine the extent of methane gene clustering and conservation. One region from the M. thermoautotrophicum strains delta H and Winter, extending approximately 13.5 kb upstream from the adjacent mvhDGAB and mrtBDGA operons that encode the methyl-viologen-reducing hydrogenase (MVH) and the methyl coenzyme M reductase II (MRII), respectively, was sequenced, and 76% sequence identity and very similar gene organizations were demonstrated. Five closely linked open reading frames were located immediately upstream of the mvh operon and were designated flpECBDA. The flpCBD genes encode amino acid sequences that are 31, 47, and 65% identical to the primary sequences of the alpha and beta subunits of formate dehydrogenase and the delta subunit of MVH, respectively. Located immediately upstream of the flp genes was the mth gene, which encodes the H2-dependent methylene-tetrahydromethanopterin dehydrogenase (MTH). In contrast to this mth-flp-mvh-mrt cluster of methane genes, a separate approximately 5.4-kb genomic fragment cloned from M. thermoautotrophicum delta H contained only one methane gene, the mtd gene, which encodes the 8-hydroxy-5-deazaflavin (H2F420)-dependent methylene-tetrahydromethanopterin dehydrogenase (MTD). Northern (RNA) blot experiments demonstrated that mth was transcribed only at early growth stages in fermentor-grown cultures of M. thermoautotrophicum delta H, whereas mtd was transcribed at later growth stages and in the stationary phase. Very similar transcription patterns have been observed by T.D. Pihl, S. Sharma, and J. N. Reeve (J. Bacteriol. 176:6384-6391, 1994) for the MRI- and MRII-encoding operons, mrtBDGA and mcrBDCGA, im M. thermoautotrophicum deltaH, suggesting coordinated regulation of methane gene expression. In contrast to the growth phase-dependent transcription of the mth/mrt and mtd/mcr genes, transcription of the mvhDGAB and frhADGB operons, which encode the two (NiFe) hydrogenases in M. thermoautotrophicum deltaH, was found to occur at all growth stages.