Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-kappaB signaling pathway. Activation of transcription factor NF-kappaB depends on the integrity of the IkappaB kinase (IKK) complex. We found that arsenite potently inhibits NF-kappaB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKalpha/beta. The affinity of IKKbeta for trivalent arsenic was verified in vitro by the ability of IKKbeta to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKbeta. Overexpression of IKKbeta (C179A) protects NF-kappaB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.