Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation mediated by yAP-1 is inducible upon exposure to several oxidants, including diamide or H2O2. We have analyzed deletion mutations that define the extreme carboxy-terminus of yAP-1 as essential for inducibility by oxidizing agents. Western blot analysis, using an antiserum that allows detection of yAP-1 expressed at single-copy levels, has shown that steady-state levels of yAP-1 or the various deletion mutant forms of this protein do not increase upon oxidative stress. A lexA-yAP-1 fusion protein was able to induce lexA-dependent reporter gene expression in response to diamide exposure but not to H2O2. Insight into the specific amino acid residues important for oxidative stress induction of yAP-1 has been obtained from an alanine scanning mutagenesis of the carboxy-terminus. Three repeats of cysteine-serine-glutamic acid have been found to be essential for normal regulation of yAP-1 by oxidants. A mutant factor lacking one of these CSE repeats produces hyper-resistance to diamide and also strongly transactivates the yAP-1 target genes GSH1 and YCF1. These data indicate that regulation of yAP-1 by oxidizing agents is a complex post-translational process that requires different regions of the factor depending on the nature of the oxidant to which the cell is exposed.