The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.