The plasmid-encoded arsenical resistance operon of plasmid pKW301 confers a high level resistance to arsenicals and antimonials on cells of Escherichia coli. Although it has been reported that ArsR of pKW301, a homologue of negative regulatory proteins, was previously shown to exhibit only weak homology to other ArsR proteins, it was identified as a DNA-binding protein by its behavior in gel mobility shift assays. In this study, ArsR exhibited a specific affinity for the ars promoter region. Gel mobility shift assay in the presence of sodium arsenite and potassium antimonial tartrate indicated that these compounds were inducers of expression of the ars operon. The specific binding site, as determined by DNase I footprint analysis, spans nucleotides 28 and 31 of the coding and noncoding strand, respectively, in the ars promoter region. ArsD, a second putative repressor protein encoded by arsD, was also identified as a DNA-binding protein by eel mobility shift assay.