Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation. Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress. Mutants which are unable to synthesis GSH due to a gene disruption in GSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions. Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, beta-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes. In addition, the GSH 1-disruption strain is sensitive to oxidative stress caused by H2O2 and tert-butyl hydroperoxide. The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions.