Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.