The effect of intracellular iron content on transferrin and iron uptake by cultured hepatocytes isolated from fetal rat liver was examined with ferric ammonium citrate and the iron chelator desferrioxamine (DFO). Incubation of the cells with ferric ammonium citrate for 24 h significantly increased the cellular nonheme iron level, whereas the number of transferrin binding sites and the uptake of transferrin and iron were reduced. In contrast, when iron-treated cells were incubated with DFO for 24 h, the cellular nonheme iron level was not altered, but the number of transferrin binding sites was increased. Treatment of the cells with exogenous iron and/or DFO did not affect the uptake of transferrin and iron by the nonsaturable processes. These results indicated that, in cultured hepatocytes, transferrin receptor expression and the subsequent uptake of transferrin and iron are regulated by the size of an intracellular, chelatable iron pool, whereas the uptake of iron by the nonsaturable processes is dependent on the extracellular transferrin concentration.