The plasmid-borne arsenical resistance (ars) operon encodes an arsenical-translocating ATPase and confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the toxic compounds from the cytosol. The trans-acting regulatory ArsR protein was shown to bind to a fragment of DNA containing the ars promoter. Hybrid formation of the ArsR protein with a ArsR-beta-lactamase chimeric protein suggested that the active form of the ArsR repressor is a dimer. From footprinting analysis the binding site was defined as a region of imperfect dyad symmetry just upstream of the -35 site. In vivo the operon was derepressed by oxyions of +III oxidation state of arsenic, antimony, and bismuth, as well as arsenate (As(V)), whereas in vitro ArsR protein-operator interaction was reduced by each of those compounds except arsenate, as determined by gel retardation and DNase I and hydroxyl radical footprinting experiments. This indicates that arsenate is not a true inducer and must be reduced to arsenite in vivo to induce. An operator mutant obtained by deletion of the in vitro ArsR-protected DNA sequence exhibited constitutive ars promoter activity, demonstrating that the binding site is the functional target for the ArsR repressor in vivo.