gamma-glutamylcysteine (gamma-GC) is an essential precursor for the biosynthesis of glutathione (GSH). The formation of gamma-GC has been considered as the rate-limiting intermediate. The purpose of this study was to enhance the gamma-GC production by a mutant strain derived from Saccharomyces cerevisiae BCRC 21727. The mutant strains were screened by the strategy including selection of methylglyoxal (MG) sensitive mutants through ethyl methanesulfonate (EMS) mutation and population analysis. The mutant's characteristics and the effects of media compositions on the cell growth and gamma-GC production were then studied. The results showed that a significantly high gamma-GC-producing mutant, SY-35679, was isolated from the MG-sensitive mutants. The intracellular gamma-GC content in the mutant SY-35679 rose to 4.77 mg(.)g(-1) DCW and had around eight folds increase, compared with that of the parent strain (0.59 mg(.)g(-1)DCW), while GSH content was at a similar level inside the mutant cell. The biosynthetic enzymes, GSH I and GSH II, were compared between the mutant and parent strain. The specific activity of GSH I in the mutant SY-35679 was found to be significantly higher than that of the parent strain, however, the GSH II specific activity maintained at a similar level. In additions, the intracellular gamma-GC and GSH contents were highly affected by different concentrations of glucose and L-cysteine supplement. The medium containing glucose of 20 g. L-1 caused the significant increase in both gamma-GC and GSH production. The proper addition of L-cysteine was also proved to have a positive effect on the levels of gamma-GC and GSH.