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Transferrin (Tf) is an iron transport protein expressed in MCF-7 human breast cancer cells. In nuclear run-on assays, 17β-estradiol (E2) increased the rate of Tf gene expression approximately 3-fold within 1 h after treatment and reporter gene activity was also induced in MCF-7 cells transfected with a construct containing a −3600 to +39 Tf gene promoter insert. Deletion and mutation analysis identified an E2-responsive promoter region between −811 and −762, which was GC-rich (80%) and contained two nonconsensus estrogen response elements (EREs). E2-responsiveness of this region was associated with a GGACA(N) 3 TGGCC motif (−803 to −791) which bound human estrogen receptor α (hERα) in gel mobility shift assays. In Drosophila Schneider SL-2 cells, the −811 to −752 was E2-responsive after cotransfection with hERα expression plasmid plus E2, whereas Sp1 protein did not induce transactivation. These studies confirm that E2 induces Tf gene expression through a nonconsensus distal ERE.