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Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in estrogen responsive tissues. Estrogen receptors α and β regulate production of VEGF in both breast and endometrial cancer cells. Alternative splicing of ER-α mRNA generates a mixture of transcripts with various exon deletions in normal breast and breast cancer cells and some of these variants are overexpressed in breast cancer. We analyzed the role of exon-deleted variants of ER-α in regulation of VEGF production by simultaneous transient transfection of CHO and MDA-MB-231 cells with a VEGF promoter luciferase construct. Estrogen (10nM) treatment resulted in a 6-fold increase in luciferase activity in cells transfected with the exon 3 deleted variant (ERΔ3) compared to a 2-fold activity induction in cells transfected with wild type ER-α. Exon 5 and exon 7 deleted variants were unable to induce activation of the VEGF promoter. Using specific deletion constructs of the VEGF promoter linked to luciferase, we showed that the majority of activation by ERΔ3 was restricted to the −70 to −88bp fragment that contains two Sp1 sites. Site-directed mutagenesis of both Sp1 sites indicated that ERΔ3 activates the VEGF promoter through interaction with Sp1 proteins. ERΔ3, a variant frequently overexpressed in breast cancer, may significantly contribute to the production of VEGF thus resulting in enhanced tumor growth in vivo.