The H+ coupled divalent metal transporter 1 (DMT1) plays a key role in iron homeostasis and is highly expressed in kidney. The aim of this study was to determine the cellular location of DMT1 in proximal tubule (PT) cells as a first step to understanding its role in the kidney. We performed RT-PCR, immunoblotting and immunostaining experiments using rat kidney cortex and the rat S1 PT derived WKPT-0293 Cl.2 cell line. mRNAs encoding all four DMT1 splice variants were detected in the tissue and the cell line by RT-PCR, and DMT1 protein was demonstrated by immunoblotting. Immunostaining showed intracellular location of DMT1 protein but no expression in the plasma membrane. DMT1 partially co-localized with the endo-/lysosomal proteins cathepsin-L and LAMP1. Immunogold labelling also detected DMT1 in the membranes of late endo-/lysosomes. Internalization of AlexaFluor®546-coupled transferrin was only observed following application to the apical membrane of PT cells. Within the cells, transferrin co-localized with DMT1. These findings have implications for renal handling of iron and nephrotoxic metals that are DMT1 ligands, such as Cd2+.