Pyocyanin (PyO-) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0-7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO(-)-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO- was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by approximately 50%) than the intact PyO-, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO- analog in which the -O- moiety was replaced by the methoxy group (-OMe), was resistant to oxidation, suggesting that oxidation of PyO- involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO- and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.