The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of a and 0i subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) a subunit precedes that of the 13.5-kDa 0i subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both a subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed ,I subunits (124 and 123 amino acid residues, respectively) share 71 % sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was (1,20), and gram-negative photosynthetic bacteria such as Rhodobacter capsulatus (8). The presence of the [NiFe] hydrogenase in these bacteria has been proven by purification and characterization of the enzymes (1,6,10,13,14,18,20,22) and, more recently, by Southern blotting, cloning, and sequencing of the genes for this hydrogenase (8,9,21