Non-transferrin-bound iron (NTBI) plays an important role in the hepatocellular injury induced by iron overload. However, the mechanism responsible for NTBI uptake into hepatocytes remains poorly defined. The purpose of this study was to define the kinetics of NTBI uptake by isolated rat hepatocytes and to characterize the uptake process. NTBI uptake was time and temperature dependent, exhibited a Michaelis-Menten constant (Km) value of 1.25 microM and maximum uptake of 241 pmol.10(6) cells-1.min-1, and 55Fe was incorporated in part into intracellular ferritin. Uptake was Ca2+ dependent, exhibiting 15 and 80% of maximal uptake in the presence of 0.6 and 0.75 mM CaCl2, respectively. The putative NTBI transporter was highly specific; divalent (Zn2+, Mn2+, Cd2+, and Co2+) or trivalent (La3+) cations did not inhibit Fe3+ uptake. Reduction from Fe3+ to Fe2+ was not essential for uptake or the process occurred deep within the membrane bilayer, since the Fe2+ chelator ferrozine did not influence 55Fe uptake. These data provide evidence for a low Km plasma membrane transporter for NTBI, which should be functional at physiological serum concentrations and saturated in iron-overload diseases, such as hemochromatosis.