Shot-wise supplementation of cysteine to a yeast culture is a common means of promoting glutathione (GSH) production. In the present work, we study the accumulation kinetics of cysteine, gamma-glutamylcysteine, and GSH and the expression of genes involved in GSH and sulfur metabolism in ethanol-stat fed-batch cultures as a result of switching to a medium enriched with cysteine and glycine. Supplementation in this fashion resulted in a rapid but short-term increase in the rate of GSH synthesis, while the expression of GSH1 decreased. Expression of GSH1 and GSH synthesis rate were observed to revert close to the base level after a few hours. These results indicate that, under such conditions, the control of GSH synthesis at higher concentrations occurred at the enzymatic, rather than the transcriptional level. The incorporation of cysteine into GSH was limited to similar to 40% of the theoretical yield, due to its requirement as a source of sulfur for protein synthesis under conditions whereby the sulfate assimilation pathway is down-regulated. This was supported by the expression profiles of genes involved in cysteine and homocysteine interconversion.