Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: alpha (approximately 2.9 kilobase pairs), and beta (approximately 2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-alpha protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-beta protein lacked the amino-terminal portion of the TfR2-alpha protein including the putative transmembrane domain. Northern blot analysis showed that the alpha transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-alpha. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound (55)Fe uptake. Taken together, TfR2-alpha may be a second transferrin receptor that can mediate cellular iron transport.