The mechanisms of uptake of non-transferrin-bound iron by human hepatoma cells (HuH7) were investigated using 59Fe-citrate and [14C]citrate. The amount of iron associated with the cells increased linearly with time, whereas citrate uptake reached a plateau after 45 min, resulting in a cellular accumulation of iron over citrate. The cells displayed high-affinity membrane binding sites for citrate with maximum binding of 118 +/- 17 pmol citrate/mg protein and a dissociation constant of 21 +/- 2 microM (n = 3). Iron uptake was saturable with a maximum uptake rate of 1.95 +/- 0.43 pmol . mg protein-1 . min-1 and an apparent Michaelis constant of 1.1 +/- 0.1 microM. Nonradioactive ferric citrate and citrate inhibited 59Fe uptake to a similar degree. This suggests that the uptake of citrate-bound iron is dependent on either binding to specific citrate binding sites or the concentration of unbound iron. The uptake of iron was inhibited by ferricyanide (>100 microM) and ferrous iron chelators but stimulated by ferrocyanide and ascorbate, suggesting that the iron is reduced from Fe3+ to Fe2+ and transported into the cell by an iron carrier-mediated step.