Effect of H2O2-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H2O2 can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H2O2 stresses with different concentrations (1 mmol/L at 4 h, 2 mmol/L at 8 h, and 4 mmol/L at 12 h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H2O2. (C) 2008 Elsevier Ltd. All rights reserved.