For more than a decade, it has been recognized that arsenate [H2AsO41-; As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster (arrAB; arsenate respiratory reduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of either arrA or arrB are incapable of growing on As(V), yet both are able to grow on a wide variety of other electron acceptors as efficiently as the wild-type. Complementation by the wild-type sequence rescues the mutants' ability to respire As(V). arrA is predicted to encode a 95.2-kDa protein with sequence motifs similar to the molybdenum containing enzymes of the dimethyl sulfoxide reductase family. arrB is predicted to encode a 25.7-kDa iron–sulfur protein. arrA and arrB comprise an operon that contains a twin arginine translocation (Tat) motif in ArrA (but not in ArrB) as well as a putative anaerobic transcription factor binding site upstream of arrA, suggesting that the respiratory As(V) reductase is exported to the periplasm via the Tat pathway and under anaerobic transcriptional control. These genes appear to define a new class of reductases that are specific for respiratory As(V) reduction.