Several Chinese hamster V79 cell line variants resistant to arsenite and one arsenite-hypersensitive variant have been isolated. The basis for the variation in arsenite sensitivity was studied by transport experiments using radiolabeled arsenite. Two arsenite-resistant variants (As/R7 and As/R27) exhibited decreased accumulation of arsenite, and the hypersensitive variant (As/S5) exhibited increased arsenite accumulation compared with the parental line. Cells depleted of endogenous energy reserves were loaded with radiolabeled arsenite, and the rate of arsenic efflux was measured. Arsenite-resistant variants exhibited an increased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with the protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase also decreased arsenite efflux, suggesting the involvement of an arsenite-glutathione complex. However, separation of the products of extrusion and the intracellular arsenic species by paper chromatography followed by autoradiography failed to show the appearance of an arsenite-glutathione complex in either case. Rather, all label in the product of the transport reaction appeared to be arsenite whether cells were loaded with arsenate or arsenite, indicating first that intracellular reduction of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was associated with high-molecular-weight material, possibly protein. Our results demonstrate the existence of an energy-dependent arsenical efflux pump in mammalian cells and show that arsenic is extruded as arsenite.