Ferrochelatase from rat liver mitochondria was purified 628-fold with a 25% yield to apparent homogeneity. The purification procedure involved solubilization of the enzyme with sodium cholate, followed by ammonium sulfate fractionation and blue Sepharose CL-6B column chromatography. The molecular weight of the enzyme was estimated to be 42,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sepharose 6B gel filtration gave a molecular weight of 240,000. The purified enzyme was analyzed for amino acid composition and shown to have abundant amounts of lysine (11%) and hydrophobic amino acid residues (48%). The enzyme was quite stable in a buffer containing 20% glycerol and 1 mM dithiothreitol. When the enzyme was assayed in the presence of palmitic acid, the specific activity for mesoheme synthesis was approximately 12,000 nmol formed/30 min/mg of protein at 37 degrees C; for protoheme synthesis, a value of 3,500 nmol formed/30 min/mg of protein was obtained. The optimum pH for the reaction was 7.8, and the Km values for the substrates were as follows: protoporphyrin IX, 28.5 microM; mesoporphyrin IX, 26.7 microM; iron with protoporphyrin IX, 33.1 microM; and iron with mesoporphyrin IX, 37.4 microM. Enzyme activity was inhibited by metals such as Co, Zn, Pb, Cu, or Mn and was highly sensitive to sulfhydryl inhibitors. The purified enzyme contained fatty acids, and its activity was markedly stimulated by their addition. Phospholipids slightly stimulated enzyme activity. Short chain carbonic acids and neutral lipids produced no effects.