Glutathione is a key player in cellular redox homeostasis and, therefore, in the response to H(2)O(2), but the factors regulating oxidation-activated glutathione synthesis are still unclear. We investigated H(2)O(2)-induced glutathione synthesis in a conditional Arabidopsis catalase-deficient mutant (cat2). Plants were grown from seed at elevated CO(2) for 5 weeks, then transferred to air in either short-day or long-day conditions. Compared to cat2 at elevated CO(2) or wild-type plants in any condition, transfer of cat2 to air in both photoperiods caused measurable oxidation of the leaf glutathione pool within hours. Oxidation continued on subsequent days and was accompanied by accumulation of glutathione. This effect was stronger in cat2 transferred to air in short days, and was not linked to appreciable increases in the extractable activities of or transcripts encoding enzymes involved in the committed pathway of glutathione synthesis. In contrast, it was accompanied by increases in serine, O-acetylserine, and cysteine. These changes in metabolites were accompanied by induction of genes encoding adenosine phosphosulfate reductase (APR), particularly APR3, as well as a specific serine acetyltransferase gene (SAT2.1) encoding a chloroplastic SAT. Marked induction of these genes was only observed in cat2 transferred to air in short-day conditions, where cysteine and glutathione accumulation was most dramatic. Unlike other SAT genes, which showed negligible induction in cat2, the relative abundance of APR and SAT2.1 transcripts was closely correlated with marker transcripts for H(2)O(2) signaling. Together, the data underline the importance of cysteine synthesis in oxidant-induced up-regulation of glutathione synthesis and suggest that the chloroplast makes an important contribution to cysteine production under these circumstances.