Plasmid-mediated resistance to arsenate, arsenite, and antimony(III) is coordinately induced by arsenate, arsenite, antimony(III), and bismuth(III). Resistance to arsenate was recently shown [Silver, S., Budd, K., Leahy, K.M., Shaw, W.V., Hammond, D., Novick, R.P., Willsky, G.R., Malamy, M.H. & Rosenberg, H. (1981) J. Bacteriol. 146, 983-996] to be due to decreased accumulation of arsenate by the induced resistant cells. We report here that decreased net uptake results from accelerated efflux of arsenate by induced plasmid-containing cells of Staphylococcus aureus and Escherichia coli. The efflux system in S. aureus was inhibited by nigericin, monensin, and proton-mobilizing uncouplers; efflux was unaffected by valinomycin. The mechanism of arsenate efflux in S. aureus was apparently not by chemiosmotic coupling to the membrane electrical potential or pH gradient. The intracellular efflux system was inhibited by low pH and mercurials (reversible by mercaptoethanol). The efflux rate was relatively independent of external pH or phosphate level and showed a sigmoidal pattern of concentration dependence.