Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.