Background/Aims: The endogenous low-molecular weight iron chelator, citrate, is considered to be an important contributor to iron transport and the liver the main site of uptake of iron citrate in subjects suffering from diseases of iron overload. Moreover, the citrate-metabolising enzyme, aconitase, is implicated in the regulation of cellular iron metabolism. This study was undertaken to determine the role of citrate and ferric citrate in the uptake of iron by rat hepatocytes.Methods: Cultured rat hepatocytes were incubated (37 degrees C, 15 min) with 100 mu M [C-14]-citrate in the presence or absence of 1.0 mu M Fe-55. Membrane-bound and intracellular radiolabel were separated by incubation with the general protease, Pronase.Results: Our results suggest that ferric citrate uptake is mediated by a specific citrate binding site which exhibits a higher affinity for citrate in the presence of iron than in its absence. Citrate was internalised by hepatocytes, with at least 70% being oxidised to CO2 within 15 min. Citrate uptake was pH-dependent, did not require the presence of sodium and increased with increasing iron concentration, Metabolic energy, anion channels, the Na+, K+-ATPase and vesicle acidification do not appear to play a role in uptake of ferric citrate, but functional sulphydryl groups may be involved.Conclusions: The data suggest either that ferric citrate complexes with higher molar ratios of iron to citrate relative to the incubation medium are bound preferentially to the membrane, or that once citrate has delivered its iron to the membrane, the complex dissociates and the components are internalised separately.