We have previously shown that hepatocytes in long-term dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 micromol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 micromol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in the total amount of ferritin. The deviation from circularity was the largest in FeSO4-treated hepatocytes, indicating that iron not properly incorporated into ferritin caused more cellular damage. We conclude that iron-loaded hepatocytes in long-term DMSO culture represent a flexible system for studying the effects of chronic iron loading on hepatocytes.