A meso-substituted cationic porphyrin (TMPyP) showed a photocytotoxicity against Gram-positive and Gram-negative bacteria. In order to determine the mechanism involved in the phototoxicity of this photosensitizer, electron paramagnetic resonance (EPR) experiments with 2,2,6,6-tetramethyl-4-piperidone (TEMP), a specific probe for singlet oxygen, and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were carried out with illuminated TMPyP. An EPR signal characteristic of TEMP-singlet oxygen (TEMPO) adduct formation was observed, which could be ascribed to singlet oxygen ((1)O(2)) generated by TMPyP photosensitization. The signal for the DMPO spin adduct of superoxide anion (DMPO-OOH) was observed in DMSO solution but not in aqueous conditions. However, an EPR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-OH) was observed in aqueous conditions. The obtained results testify a primary hydroxyl radical ((center dot)OH) generation probably from superoxide anion (O(2)(center dot-)) via the Fenton reaction and/or via Haber-Weiss reaction. Gram-positive and Gram-negative bacteria inactivation by TMPyP photosensitization predominantly involved Type II reactions mediated by the formation of (1)O(2), as demonstrated by the effect of quenchers for (1)O(2) and scavengers for (center dot)OH (sodium azide, thiourea, and dimethylsulphoxide). Participation of other active oxygen species cannot however be neglected since Type I reactions also had a significant effect, particularly for Gram-negative bacteria. For Gram-negative bacteria the photoinactivation rate was lower in the presence of superoxide dismutase, a specific O(2)(center dot-) scavenger, and/or catalase, an enzyme which specifically eliminates H(2)O(2), but was unchanged for Gram-positive bacteria. The generation of (1)O(2), O(2)(center dot-) and (center dot)OH by TMPyP photosensitization indicated that TMPyP maintained a photodynamic activity in terms of Type I and Type II mechanisms. (C) 2008 Elsevier Ltd. All rights reserved.