A solubilized protein with ferrochelatase activity has been extracted from hepatic mitochondria of Sprague-Dawley rats. Under anerobic conditions in the presence of sodium ascorbate the ferrochelatase velocity was typically 1.8 nM X min-1 X mg-1. The extract also displayed zinc chelatase activity of 1.2 nM X min-1 X mg-1 without either anerobic conditions or ascorbate. In both cases substrate inhibition occurred for metal ion and deuteroporphyrin, but in the linear range a noncompetitive two-site mechanism was observed. The ferrochelatase activity is inhibited by divalent copper, mercury, and lead ions and the sodium salt of p-chloromercuribenzoate and is replaced by zinc chelation activity. This evidence suggests that the metal-binding site includes a thiol group. The inhibition of the site is greatest with Cu2+ and decreases with increasing ionic radius to Pb2+. This observation implies that the binding site is stereochemically adapted to the small Fe2+ ion and to some extent protected from larger, sulfur-binding ions which can inhibit ferrochelatase activity.