The uptake of oxidized glutathione (GSSG) into inside-out membrane vesicles of Wistar rat erythrocytes was studied. Uptake was ATP dependent, into an osmotically active space, and saturable. Analysis of saturable ATP-dependent GSSG uptake showed two affinities for GSSG [concentration for half-maximal velocity (K1/2 1), 26 microM; K 1/2 2, 4 mM; maximum transport rate (Vmax 1), 100 pmol.mg-1.min-1; Vmax 2, 360 pmol.mg-1.min-1]. Interactions of the high-affinity system with different organic compounds were studied. Leukotriene C4, bromosulfophthalein-S-glutathione, and 2,4-dinitrophenyl-S-glutathione were effective inhibitors. In addition, anionic nonglutathione conjugates, like indocyanine green, rose bengal, dibromosulfophthalein, and sulfated or glucuronidated (divalent) bile acids inhibited GSSG transport. Monovalent bile acids had no influence on GSSG transport. Inhibition by 2,4-dinitrophenyl-S-glutathione [inhibition constant (Ki) = 2.6 microM] and sulfated glycolithocholic acid (Ki = 2.9 microM) was purely competitive. The use of adenosinetriphosphatase (ATPase) inhibitors suggested a resemblance with E1E2-type ATPase. Vesicles of erythrocytes isolated from the TR- rat, a mutant rat strain with a defective biliary secretion of organic anions, have an impaired uptake of GSSG (Vmax was decreased 2-fold). In conclusion, erythrocytes have an ATP-dependent organic anion transport system that can be inhibited by a broad range of organic anions. This system is very similar if not identical to the hepatocanalicular ATP-dependent organic anion transporter.