In vitro studies with freshly drawn human erythrocytes showed 4-dimethylaminophenol, a cyanide antidote, to be rapidly metabolized with the formation of a transient S,S-(2-dimethylamino-5-hydroxy-1,3-phenylene)bis-glutathione conjugate and a stable S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)tris-glutathione conjugate. The stable tri-glutathionyl derivative was actively transported across the red cell membrane with an apparent Vmax = 1 nmol/min/ml red cell suspension (15 g hemoglobin/100 ml) and Km = 0.5 mM. The transport system was strictly unidirectional, inhibited completely by sodium fluoride and reduced to one-fifth by lowering the temperature from 37 to 22 degrees. Similarly S-(2,4-dinitrophenyl)-glutathione, the glutathione-S-transferase mediated glutathione-S-conjugate with 1-chloro-2,4-dinitrobenzene, was unidirectionally transported, a process which was inhibited by sodium fluoride. Kinetic analysis revealed two different transport processes: Vmax = 0.9 nmol/min/ml, Km = 1.4 microM and Vmax = 4.5 nmol/min/ml, Km = 700 microM. Mutual inhibition of the low affinity transport system was found for both glutathione-S-conjugates. The apparent energies of activation for all these transport processes and for GSSG were identical (70 kJ/mol) suggesting at least one common carrier for the excretion of the three glutathione-S-conjugates.