Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N- and C-terminal nucleotide-binding domains connected by a 25-residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the 5-residue insertion had wild-type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type enzyme. Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.