Gamma-glutamylcysteine (gamma-GC), the precursor of glutathione (GSH), may have significant health benefits as a dietary supplement, but there are few cost-effective methods available for its large-scale production. We developed an efficient method for producing gamma-GC in a mutant yeast strain using a three-step breeding procedure and a unique cultivation process. In the first breeding step, we prepared a glutathione synthetase (GSH2)-deficient yeast mutant. In the second step, selenate (SeO42-) sensitivity was introduced by crossing the GSH2-deficient mutant with a strain harboring the met30 mutation. In the final step, pantothenic acid auxotrophy was introduced by ethyl methanesulfonate mutagenesis. The isolated strain displayed significantly enhanced cellular gamma-GC when cultivated in synthetic medium without pantothenic acid, reaching a maximum level of 4.39% of dry cell weight. Using this strain, we were able to prepare a yeast extract containing approximately 13% gamma-GC (w/w), which is markedly higher than the reported value (0.3%) of commercially available yeast extracts. The present method may facilitate large-scale gamma-GC production for investigating the nutritive value and other benefits of dietary gamma-GC. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.