Hephaestin is a multicopper ferroxidase involved in iron absorption in the small intestine. Expressed mainly on the basolateral surface of duodenal enterocytes, hephaestin facilitates the export of iron from the intestinal epithelium into blood by oxidizing Fe(2+) into Fe(3+), the only form of iron bound by the plasma protein transferrin. Structurally, the human hephaestin ectodomain is predicted to resemble ceruloplasmin, the major multicopper oxidase in blood. In addition to its ferroxidase activity, ceruloplasmin was reported to oxidize a wide range of organic compounds including a group of physiologically relevant substrates (biogenic amines). To study oxidation of organic substrates, the human hephaestin ectodomain was expressed in Pichia pastoris. The purified recombinant hephaestin has an average copper content of 4.2 copper atoms per molecule. The K(m) for Fe(2+) of hephaestin was determined to be 3.2μM which is consistent with the K(m) values for other multicopper ferroxidases. In addition, the K(m) values of hephaestin for such organic substrates as p-phenylenediamine and o-dianisidine are close to values determined for ceruloplasmin. However, in contrast to ceruloplasmin, hephaestin was incapable of direct oxidation of adrenaline and dopamine implying a difference in biological substrate specificities between these two homologous ferroxidases.