Microcytic anemia (mk) mice and Belgrade (b) rats have severe iron deficiency anemia due to defects in intestinal iron transport and erythroid iron utilization. Both animal mutants carry the same missense mutation in Nramp2, the first mammalian iron transporter to be identified. This mutation, in which glycine 185 is changed to arginine (G185R), occurs within predicted transmembrane domain 4 of the protein. We have performed site-directed mutagenesis of murine Nramp2, focusing on amino acids of transmembrane domain 4 that are highly conserved among Nramp-like proteins. We have expressed each mutant form in transfected cells and examined iron transport function, subcellular localization, and protein amounts. All tested forms of Nramp2 localize to the plasma membrane and to transferrin-containing endosomes. Most transmembrane domain 4 mutations affect the amount of protein detected and consequently show diminished iron transport. The G185R mutation, however, causes near total loss of Nramp2 function that cannot be fully explained by a decreased amount of protein, indicating that G185R disrupts iron transport through an alteration in the function of Nramp2, rather than degradation of the protein.