ABSTRACT In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent metal transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split‐ubiquitin yeast 2‐hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6‐kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage‐dependent anion‐selective channel protein‐1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the metal into the mitochondria.—Wolff, N. A., Ghio, A. J., Garrick, L. M., Garrick, M. D., Zhao, L., Fenton, R. A., Thévenod, F. Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1). FASEB J . 28, 2134–2145 (2014). www.fasebj.org