Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (ΔNT), carboxyl terminus (ΔCT), as well as in NPAY^28-31, YSCF^62-65, and YLLNT^555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK₁ kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (ΔCT and ΔYLLNT) had an increased fraction of the “recycling pool” that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the ΔYLLNT and ΔCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (−IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.