The tripeptide gamma-L-glutamyl-L-cysteinylglycine (glutathione) is one of the major antioxidant molecules of cells and plays a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. In the yeast Saccharomyces cerevisiae, the first enzyme involved in glutathione biosynthesis, gamma-glutamylcysteine synthetase, is encoded by the GSH1 gene. This study shows that the regulation of the yeast GSH1 gene by oxidants and the heavy metal cadmium is at the level of transcription. We also demonstrate that the regulation of the GSH1 gene by H2O2 depends upon the presence of the amino acids glutamate, glutamine and lysine in the media. Moreover, regulation of the GSH1 gene by H2O2, although requiring the Yap1 protein, appears to be mediated by a mechanism distinct from that which regulates the Yap1-dependent induction of genes encoding thioredoxin (TRX2) and a stress-inducible HSP70 (SSA1) by H2O2.