The liver is one of the principal sites of iron overload in diseases such as hemochromatosis and beta thalassemia. Hence, much effort has been invested in examining the mechanisms of Fe uptake by hepatocytes. In the present study we have examined the effect of small molecular weight (M-r) Fe complexes on Fe uptake from iron 59-labeled transferrin (Tf) and Fe-59-labeled citrate by primary cultures of hepatocytes, This was important to assess because Fe-citrate and saturated diferric Tf coexist in the serum of patients with untreated Fe overload. Preincubation of hepatocytes with the low-M-r Fe complex ferric ammonium citrate (FAC; 25 mu g/ml; (Fe) = 4.4 mu g/mL) followed by incubation with Fe-59-Tf or Fe-59-citrate ((Fe) = 0.25 to 25 mu mol/L) resulted in the marked stimulation of Fe-59 uptake. For example, at a physiologically relevant Tf-Fe concentration of 25 mu mol/L, there was an 8-fold increase in 59Fe uptake by cells incubated with FAC compared to control cells. In contrast, at Tf-Fe concentrations of 0.25 to 2.5 mu mol/L, 59Fe uptake in FAC-treated cells was only 1-fold to 3-fold greater than that in the corresponding controls. These data suggest that the FAC-activated Fe uptake process predominates at physiologically relevant Tf concentrations above the saturation of the Tf receptor (TfR). This is the first study to demonstrate that preincubation of hepatocytes with low-M-r Fe complexes can markedly increase Fe uptake from diferric Tf. In conclusion, these results may help to explain the loading of hepatocytes with Fe that occurs in Fe-overload disease despite marked down-regulation of the MR.