Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is coenzyme F420-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L. (1989) Can. J. Microbiol. 35, 499-507), and the other (MTH) is coenzyme F420-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R. K. (1990) FEBS Lett. 261, 112-116). Based on the NH2-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M. thermoautotrophicum DNA. Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on coenzyme F420, thermostable, thermophilic, and exhibited maximum activity at an acidic pH. The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R. K., and Klein, A. (1991) Eur. J. Biochem. 202, 1205-1208), but comparisons with coenzyme F420 utilizing enzymes revealed a conserved region at the NH2 terminus of MTD that could correspond to the ability to interact with coenzyme F420. The mtd transcript was approximately 900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence. The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.