AIM To study the role of protoporphyrin IX (pPIX) in mitochondrial metabolism of hydrogen peroxide (H2O2). METHODS O2 (-) specific fluorescent markers DMA (9,10-dimerthylanthracence) and SOSG (Singlet Oxygen Sensor Green reagent) were used for measurement of singlet oxygen ((1)O2). Catalyzing conversion of H2O2 into (1)O2 by pPIX was monitored in vitro under varied H2O2 content, temperature, and PH value in the reaction. Ex vivo mitochondrial model was used to analyze effects of ferrochelatase (FECH) and high energy X-rays on this catalytic reaction. RESULTS In complete dark, measurable (1)O2 was generated when 1.5 mM of H2O2 was incubated with 24 μM of pPIX H2O2 at 37°C for 3 hours. Mitochondrial yield of H2O2 was 0.11±0.03 nmole/mg/min. Mitochondrial FECH significantly improve the catalytic ability of pPIX converting H2O2 into (1)O2. At presence of high-energy X-ray, incubation of 14.4 μM of pPIX with 0.54 μM of H2O2 also generated (1)O2, during which the fluorescence density of 1.05 μM of DMA decreased by 41.5% (P < 0.05). This conversion was not observed when pPIX was replaced with structurally similar hematoporphyrin. CONCLUSION pPIX can catalyze conversion of H2O2 into (1)O2.