We recently identified a novel factor involved in cellular iron assimilation called SFT or Stimulator of Fe Transport (Gutierrez, J. A., Yu, J., Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol. 149, 895-905). When stably expressed in HeLa cells, SFT was found to stimulate the uptake of both transferrin- and nontransferrin-bound Fe (iron). Assimilation of nontransferrin-bound Fe by HeLa cells stably expressing SFT was time- and temperature-dependent; both the rate and extent of uptake was enhanced relative to the activity of control nontransfected cells. Although the apparent Km for Fe uptake was unaffected by expression of SFT (5.6 versus 5.1 microM measured for control), the Vmax of transport was increased from 7.0 to 14.7 pmol/min/mg protein. Transport mediated by SFT was inhibitable by diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and Fe2+-specific chelators. Because cellular copper status is known to influence Fe assimilation, we investigated the effects of Cu (copper) depletion on SFT function. After 4 days of culture in Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was reduced by more than 60%. Both control cells and cells stably expressing SFT displayed reduced Fe uptake as well; levels of transferrin-mediated import fell by approximately 80%, whereas levels of nontransferrin-bound Fe uptake were approximately 50% that of Cu-replete cells. The failure of SFT expression to stimulate Fe uptake above basal levels in Cu-depleted cells suggests a critical role for Cu in SFT function. A current model for both transferrin- and nontransferrin-bound Fe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the divalent cation across the membrane bilayer. SFT expression did not enhance levels of HeLa cell surface reductase activity; however, Cu depletion was found to reduce endogenous activity by 60%, suggesting impaired ferrireductase function may account for the influence of Cu depletion on SFT-mediated Fe uptake. To test this hypothesis, the ability of SFT to directly mediate Fe2+ import was examined. Although expression of SFT enhanced Fe2+ uptake by HeLa cells, Cu depletion did not significantly reduce this activity. Thus, we conclude that a ferrireductase activity is required for SFT function in Fe3+ transport and that Cu depletion reduces cellular iron assimilation by affecting this activity.