Iron, as ferric nitrilotriacetate or ferric ammonium citrate, was administered to rat hepatoma cells (H4AZC2) that were grown in serum-containing media or in hormone-supplemented defined media on collagen matrices. High levels of iron either retarded growth or were cytotoxic, so conditions were established for maximum iron loading where cells survived at near normal growth rates. In all cases, cells exposed to iron produced more ferritin than those grown in its absence, and elevated ferritin levels were paralleled by higher intracellular iron contents. Cells grown in serum-free media, however, took up iron more rapidly than corresponding cells in serum-supplemented media, and intracellular iron and ferritin also reached much higher levels. During exponential growth stages, for example, ferritin levels in iron-stimulated cells were 35-fold greater than those in control cells. These results indicate that transferrin is not required as an iron donor as the inorganic iron was taken up effectively and utilized to stimulate ferritin synthesis. Twenty-four hours after iron administration, endocytotic mechanisms were evident by the appearance of coated vesicles and pits and visible cytoskeletal structures. Subsequently, clusters of iron micelles appeared in the cell. Ferritin isolated from iron-overloaded cells were rich in L-type subunits, but newly synthesized ferritins in iron-stimulated or control cells had almost equivalent amounts of heavy and light subunit constituents.