Arsenic is a ubiquitous contaminant of drinking water and food. The mechanisms of the toxic action of inorganic arsenic are unknown. We report the isolation of proteins having a high affinity for arsenic in the +3 oxidation state that are induced by arsenite (AsIII) in human lymphoblastoid cells. The arsenic-binding proteins were isolated using a p-aminophenylarsine oxide affinity column. At least four proteins of 50, 42, 38.5 and 19.5 kDa were isolated by elution with 10 or 100 mM 2-mercaptoethanol. Two proteins were tentatively identified as tubulin and actin on the basis of their molecular weights and previously reported affinity for the arsenic column. The identities of the remaining proteins are unknown. Heme oxygenase 1 was induced by AsIII but did not bind to the arsenic affinity column. We conclude that AsIII induces multiple proteins that have variable affinities for arsenic in the +3 state as judged by the concentration of 2-mercaptoethanol required for their elution. The arsenic binding motif of these proteins may involve three thiol groups arranged 3-6 A apart by the tertiary structure of the protein as suggested by others. These proteins may serve as high affinity binding sites for AsIII and may be involved in the biological action of AsIII.