The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.