The structural genes for the arsenical pump of the conjugative R-factor R773 contained on a HindIII fragment of 4.3 kilobase pairs were cloned into bacteriophage M13. A series of ordered deletions was created using Bal31 digestion, and the nucleotide sequence of the operon determined. Three open reading frames for genes arsA, arsB, and arsC were found. The arsA gene encodes a hydrophilic protein of 63,169 Da with two potential adenylate-binding sites. The arsB gene encodes a potentially membrane protein of 45,577 Da. The arsC gene encodes a 15,811-Da hydrophilic protein. The arsA and arsC gene products correspond to cytosolic proteins previously identified from minicell experiments. Isolated ArsA protein was shown to bind to dye-agarose columns which act as affinity resins for nucleotide-binding proteins. A model is proposed in which these gene products form an anion translocating ATPase for extrusion of arsenite and arsenate from resistant cells.