Pyocyanin is a redox active virulence factor produced by the human pathogen Pseudomonas aeruginosa. Treatment of endothelial cells with pyocyanin (1-50 microM) resulted in the dose-dependent formation of hydrogen peroxide that was detected in the extracellular medium. Total intracellular glutathione levels decreased in response to pyocyanin in a dose-dependent manner from a control value of 19.9 +/- 2.7 nmol/mg protein to 10.0 +/- 2.4 nmol/mg protein. Prior treatment of cells with catalase afforded complete protection against loss of glutathione. Total intracellular soluble thiols decreased from 95.0 +/- 6.2 nmol/mg protein to 78.6 +/- 2.3 nmol/mg protein at the highest test dose. Intracellular levels of NADPH increased up to 2.4-fold in response to pyocyanin exposure. It is concluded that pyocyanin exposes endothelial cells to oxidative stress by the generation of hydrogen peroxide, which subsequently depletes intracellular glutathione and increases intracellular levels of mixed disulfides.