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Isolated rat hepatocytes containing 0.56–1.79 μg iron/10⁶ cells and with an intracellular ATP concentration of 3–4 mM, accumulate iron from transferrin linearly with time for at least 3 h. At 37°C the rate of uptake amounts to 0.3–0.7 pmol/mg cell protein per min. The uptake reaches a saturation level of 21–40 pmol/mg cell protein per h at 2.2 μM iron. At 5°C the uptake does not increase over the time of incubation. Uptake of iron, but not binding of transferrin is increased 4–5-fold at oxygen concentrations 10–20 μM. At oxygen concentrations beyond these limits iron uptake is decreased. Iron taken up at low oxygen concentrations can be chelated by bathophenanthroline and bathophenanthroline disulphonate, but only if the chelators are present during the uptake experiments. The results suggest that iron uptake from transferrin by hepatocytes in suspension involves reductive removal of iron.