Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: gamma-Glu-Cys-Gly), they have the general structure (gamma-Glu-Cys)nGly, where n is 2-11. In order to study the biosynthesis of phytochelatins, we used the mutagen N-methyl-N'-nitro-N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2-15% of the wild-type GSH level. For three mutants a lowered activity of gamma-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of gamma-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.