Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ƊgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ƊgshB mutants were isolated and characterized. The ƊgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ƊgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ƊgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ƊgshB strain, respectively, when cells are starved for nitrate or sulfate.