The Escherichia coli NikR transcription factor is a Ni(II)-dependent repressor that regulates the production of a nickel ion transporter. The X-ray crystal structure of the Ni(II)-NikR-DNA bound complex revealed a K+-binding site positioned at the interface of the metal-and DNA-binding domains, but the significance of the potassium was unclear. Mutation of one of the K+ ligands impairs the affinity and specificity of DNA binding in the presence of either stoichiometric or excess Ni(II). Removal of K+ abrogates Ni(II)-responsive DNA binding completely while the addition of K+ restores this activity. Furthermore, the observed K+ dependence can be relieved by replacing the K+ ligand Asp34 with an arginine. These mutagenesis and cation exchange experiments reveal that K+ is a critical structural component for the activation of Ni(II)-responsive DNA binding by NikR.