Recent studies have demonstrated that preincubation of SK-Mel-28 melanoma cells with ferric ammonium citrate (FAC) resulted in marked stimulation of ⁵⁹Fe uptake from ⁵⁹Fe-¹²⁵I-transferrin (Tf), but only at Tf concentrations above that required for saturation of the Tf receptor (Richardson and Baker (1992) J. Biol. Chem. 267, 13972–13979). The mechanism responsible for this stimulation was unknown and is the subject of the present report. Preincubation of cells with FAC (25 μg/ml), followed by a 2 h incubation with ⁵⁹Fe-¹²⁵I-Tf (0.1 mg/ml; 1.25 μM), resulted in temperature-dependent ⁵⁹Fe uptake to approx. 200% of the control value. Furthermore, the effect was not specific for melanoma cells and was also observed in other normal and neoplastic cells. Preincubation of melanoma cells with FAC also stimulated ⁵⁹Fe uptake from ⁵⁹Fe-citrate, but to a far greater extent than that observed with ⁵⁹Fe-¹²⁵I-Tf (viz., > 20-fold that seen for the control). Interestingly, neither receptor-mediated endocytosis nor the postulated diferric Tf reductase were involved in the FAC-activated Fe uptake process from Tf. However, the addition of free radical scavengers to FAC such as catalase, superoxide dismutase, ceruloplasmin, Hepes, mannitol and high concentrations of BSA or ascorbate, markedly depressed FAC-activated ⁵⁹Fe uptake from ⁵⁹Fe-¹²⁵I-Tf and ⁵⁹Fe-citrate. These agents when added to control cells had no effect on ⁵⁹Fe uptake. The addition of superoxide generating agents and hydrogen peroxide to minimum essential medium (MEM) containing FAC but not to MEM alone, also stimulated ⁵⁹Fe uptake. These data suggest that the initial activation of the FAC-stimulated Fe uptake system was caused by the production of hydroxyl radicals via the Fe-catalysed Haber-Weiss reaction. We propose that this Fe uptake process represents an important cellular defense mechanism against oxidant stress generated in the presence of low-molecular-weight Fe complexes.