The formation of zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PP) in porcine heart mitochondria was investigated by fluorescent spectral analysis. The time-course study at 37°C and pH 5.5 with 100μM ZnCl2 showed a linear formation of ZnPP from oxymyoglobin after a lag-period (9h) at the formation rate of 0.46nmol/h. The formed amount of ZnPP is dependent upon the ZnCl2 concentration. In the incubation without ZnCl2, the new peak derived from PP appeared at 633nm after 12h, and then its intensity gradually increased. The formation of PP is believed to be due to the release of Fe2+ in porphyrin ring of oxymyoglobin during the incubation. This suggests that the mitochondria have the ability to form PP from oxymyoglobin, and therefore, are directly related to the release of Fe2+ from porphyrin ring in myoglobin.