The Schizosaccharomyces pombe hmt1 gene encodes an ABC (ATP-binding cassette)-type protein essential for Cd2+ tolerance. Immunoblot analysis of subcellular fractions indicates that the native HMT1 polypeptide is associated with the vacuolar membrane. Vacuolar membrane vesicles were purified from strains that hyperproduce, or are deficient in, the HMT1 protein. In vitro transport of radiolabeled substrates by these vesicles indicates that HMT1 is an ATP-dependent transporter of phytochelatins, the metal-chelating peptides involved in heavy metal tolerance of plants and certain fungi. Vacuolar vesicles containing HMT1 are capable of taking up both apo-phytochelatins and phytochelatin-Cd2+ complexes. HMT1 activity is sensitive to antibodies directed against this protein and to vanadate, but not to inhibitors affecting the vacuolar proton ATPase or ionophores that abolish the pH gradient across the vacuolar membrane. Vacuolar uptake of Cd2+ and of a glutathione conjugate were also observed, but are not attributable to HMT1. These studies highlight the importance of the yeast vacuole in detoxification of xenobiotics.