The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472-1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E. , and Sly, W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198-13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.