Background/Aims: In hereditary hemochromatosis, iron-loading of hepatocytes is associated with increased iron uptake while little is known about iron release. This study aims to characterise iron release and ferroportin expression by Hfe knockout hepatocytes to determine if they contribute to iron overload in haemochromatosis.Methods: Iron release by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice was measured after incubation with nontransferrin-bound iron as iron-citrate.Results: Iron release and ferroportin expression by hepatocytes from Hfe knockout, non-iron-loaded and in vivo iron-loaded wild-type mice were similar although, nontransferrin-bound iron uptake was significantly increased in Hfe knockout hepatocytes and decreased in iron-loaded wild-type hepatocytes compared with non-iron-loaded wild-type cells. When expressed as a percentage of total iron uptake, iron release was decreased in Hfe knockout hepatocytes (4.6 +/- 0.7 versus 13.7 +/- 1.2%, P < 0.0001) and increased in iron-loaded wild-type hepatocytes (29.5 +/- 0.5 versus 13.5 +/- 0.7%; P < 0.0001) compared with wild-type hepatocytes. In contrast, in vitro iron-loading increased iron release and ferroportin expression by both Hfe knockout and wild-type hepatocytes.Conclusions: Hfe knockout hepatocytes accumulate iron as a result of limited iron export and enhanced iron uptake. The correlation between iron release and ferroportin expression suggests that iron export in hepatocytes is mediated by ferroportin. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.