Redox reactions involving cysteine thiol-disulfide exchange are crucial for sensing intracellular levels of H2O2. However, oxidation-sensitive dithiols are also sensitive to intracellular reducing agents, and disulfide bonds are thus transient. The yeast transcription factor Yap1 is activated by disulfide-induced structural changes in the nuclear export signal in a carboxy-terminal domain. We show herein that the activation of Yap1 by H2O2 requires multistep formation of disulfide bonds. One disulfide bond forms within 15 s in an amino-terminal domain, and then disulfide bonds linking the two domains accumulate. The multiple interdomain disulfide bonds, which result in reduction-resistant Yap1, are required for transduction of the H2O2 stress signal to induce the appropriate level and duration of specific transcription. Our results suggest both a mechanism wherein the H2O2 levels might be sensed by Yap1 and the way in which the NADPH levels might be maintained by altering the redox status of Yap1.