Adenosine deaminase (ADA) gene expression is induced by 17β-estradiol (E2) in MCF-7 human breast cancer cells, whereas the antiestrogens 4′-hydroxytamoxifen and ICI 182,780 exhibit partial estrogen receptor (ER) agonist/antagonist and antagonist activities, respectively. Previous studies have shown that the −211 to +11 region of the ADA gene promoter contains six GC-rich sites (I–VI) that bind Sp1 protein, and these elements are required for high basal expression. In transient transfection studies with pADA211, which contains the− 211 to +11 ADA gene promoter linked to a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, E2 and tamoxifen (but not ICI 182,780) induced CAT activity. Ligand-induced transactivation was observed only in cells cotransfected with expression plasmids for wild-type ER or HE11, which does not contain the DNA-binding domain of the ER. Cotransfection with HE15 and HE19, which contain the DNA-binding domain and activation function-1 (AF-1) and AF-2 of the ER, respectively, did not result in E2-induced activity. Subsequent deletion analysis of the ADA gene promoter showed that Sp1 binding site IV (−79 to −73) was primarily responsible for hormone responsiveness. ER activation of ADA gene expression is another example of an E2-induced gene that is dependent on ER/Sp1 interactions with a site-specific GC-rich motif.