Non-transferrin-bound iron (NTBI) uptake occurs in a variety of cells by a saturable, specific and temperature-sensitive process. Our previous studies indicated that NTBI uptake by cardiac myocytes and Hep G2 cells was reversibly up-regulated by iron deposition. In the present work we have characterized this up-regulation and examined its mechanism by comparing the uptake of oxidized (Fe3+) and ascorbate-reduced (Fe2+) forms of iron. Iron loading markedly enhanced the uptake of iron both in the presence and absence of ascorbate, but the increment was greater when ascorbate was absent. This up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein synthesis. Uptake by the iron-loaded cells was less sensitive to thiol-alkylating agents and competing metal ions, but was more sensitive to proteolysis. Iron loading causes an increase in both Km and Vmax for uptake of both Fe2+ and Fe3+, although the values differ, suggesting distinct rate-limiting steps for uptake of Fe2+ and Fe3+. Consistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing metal ions and monensin. The Fe(2+)-specific chelators bathophenanthroline disulfonate and ferrozine markedly inhibited iron uptake whether ascorbate was present or not, indicating that Fe3+ uptake is dependent on reduction to the ferrous state. This requirement for reduction was independent of the iron status of the cells, demonstrating that the process of up-regulation is not due to the appearance of a new mechanism for translocation of Fe3+ without reduction. Taken together, the evidence favors a model of NTBI transport where an obligate and rate-determining reduction of Fe3+ occurs prior to or during uptake, followed by translocation through an Fe2+ carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate suggest that exogenous Fe2+ does not access the carrier available to the nascent ferrous ion derived from the reductase and is consistent with close coupling between the reduction and the translocation processes. In iron-loaded cells with increased rates of NTBI transport, a similar mechanism prevails.