The hepatic uptake of transferrin-bound iron by a nontransferrin receptor (NTR)-mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)-mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild-type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100-fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild-type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR-mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR-mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100-fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR-mediated process entailed transferrin adsorption to membrane-bound proteins, internalization, and release of iron from transferrin by a pH-dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier-mediated mechanism.