Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/-IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/-IRE construct. (64)Cu(1+) incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. (64)Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein(-1) min(-1) and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/-IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu(1+).