Transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and a fluorescent glutathione S-conjugate, bimane-S-glutathione (B-SG) was studied in the baker's yeasts (S. cerevisiae). Both conjugates were exported from the cells; the transport was inhibited by fluoride and vanadate like in mammalian cells. B-SG was also found to be accumulated in the vacuoles. The transport rate of DNP-SG outside the cell was higher in a vacuolar-deficient strain. A significant ATP-dependent uptake of (3H)-DNP-SG by vacuoles was found. These results indicate thatS. cerevisiaetransport glutathione S-conjugates both outside the cells and into the vacuoles.