The cdhA and cdhB genes that code for the large and the small subunits of carbon monoxide dehydrogenase (CDH), respectively, were isolated from a genomic library of Methanothrix soehngenii DNA in Escherichia coli, using polyclonal antibodies raised against purified CDH. After introduction in E. coli or Desulfovibrio vulgaris, the cdh genes appeared to be expressed irrespective of their orientation, yielding immunoreactive proteins of 79 and 19 kDa, corresponding in size to the known subunits of purified CDH. However, no CDH activity could be detected in these heterologous hosts. The cdh genes are preceded by consensus ribosome-binding sites and are arranged in an operon-like structure, with cdhA preceding cdhB. Upstream from this operon, sequences similar to archaeal promoters were identified. The amino acid sequence, deduced from the primary sequence of cdhA, showed homology with ferredoxins and with acyl-CoA oxidase. This is compatible with the proposed functions of CDH.