Ferrochelatase catalyzes the insertion of ferrous ions into protoporphyrin IX to produce heme. Previously, it was found that this enzyme also participates in the reverse reaction of iron removal from heme. To clarify the role of the reverse reaction of ferrochelatase in cells, mouse liver mitochondria were fractionated to examine the localization of ferrochelatase, and it was found that the enzyme localizes not only to the inner membrane, but also to the outer membrane. Observations by immunoelectron microscopy confirmed the dual localization of ferrochelatase in ferrochelatase-expressing human embryonic kidney cells and mouse liver mitochondria. The conventional (zinc-insertion) activities of the enzyme in the inner and outer membranes were similar, whereas the iron-removal activity was high in the outer membrane. 2D gel analysis revealed that two types of the enzyme with different isoelectric points were present in mitochondria, and the acidic form, which was enriched in the outer membrane, was found to be phosphorylated. Mutation of human ferrochelatase showed that serine residues at positions 130 and 303 were phosphorylated, and serine at position 130 may be involved in the balance of the reversible catalytic reaction. When mouse erythroleukemia cells were treated with 12-O-tetradecanoyl-phorbol 13-acetate, an activator of protein kinase C, or hemin, phospho-ferrochelatase levels increased, with a concomitant decrease in zinc-insertion activity and a slight increase in iron-removal activity. These results suggest that ferrochelatase localizes to both the mitochondrial outer and inner membranes and that the change in the equilibrium position of the forward and reverse activities may be regulated by the phosphorylation of ferrochelatase.