Hepatomegaly is commonly observed in hepatic iron overload due to human hemochromatosis and in animal models of iron loading, but the mechanisms underlying liver enlargement in these conditions have received scant attention. In this study, male rats were treated with iron dextran or dextran alone for 6 months. Chronic iron loading resulted in a > 50-fold increase in hepatic iron concentration. Both liver weights and liver/body weight ratios were increased approximately 2-fold in the iron-loaded rats (P < 0.001 for both). Hepatocyte nuclei expressing proliferating cell nuclear antigen (PCNA), a marker of S phase, were significantly increased in the iron-loaded livers, suggesting enhanced proliferation. To assess the mechanisms by which iron promotes proliferation, the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, hepatocyte growth factor (HGF), and transforming growth factor-alpha (TGF-alpha) were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Of these growth-associated factors, only TNF-alpha messenger RNA (mRNA) was significantly increased by iron loading (about 3-fold; P = 0.005). Because cyclin D1 is required for entry of hepatocytes into the cell cycle after partial hepatectomy or treatment with direct mitogens, levels of immunoreactive cyclin D1 were examined and found to be significantly increased in the iron-loaded livers. The increase in cyclin D1 protein in the iron-loaded livers was paralleled by an increase in the abundance of its transcript as measured by real-time PCR. Taken together, these results suggest that iron is a direct mitogen in the liver and raise the possibility that chronic stimulation of hepatocyte proliferation may play a role in the pathophysiology of iron overload states.