The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter (Pgt1) by a genetic strategy that exploited the requirement of the cys1aDelta strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1aDelta strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione (K(m)=63 microM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu-cys-gly, an analogue of glutathione (gamma-glu-cys-gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1(+) expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.