In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe-4S](1+/0) center and at least four [4Fe-4S](2+/1+) centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.