Retinoic acid receptor alpha 1 (RAR alpha 1) gene expression is induced by 17 beta-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cells, and the -100 to -49 region of the RAR alpha 1 gene promoter was previously shown to be required for E2-responsiveness. This region of the RAR alpha 1 promoter was further analyzed using the following oligonucleotides: -100 to -49 (RAR4); -79 to -56 (RAR3); -79 to -49 (RAR2); -100 to -58 (RAR1); and their derived promoter reporter constructs (pRAR4, pRAR3, pRAR2, and pRAR1). In transient transfection studies in MCF-7 human breast cancer cells, pRAR2 and pRAR1 were E2-responsive; both of the RAR alpha 1 gene promoter inserts contained two GC-rich sites and bound Sp1 protein in gel mobility shift assays. Using wild-type [32P]RAR2 and oligonucleotides mutated in one or both GC-rich sites, it was shown that ER enhanced Sp1 binding to both sites, but a ternary ER-Sp1-DNA complex was not observed in gel mobility shift assays. In transient transfection assays, each of the GC-rich motifs were sufficient for E2-induced transactivation. In ER-negative MDA-MB-231 cells transiently transfected with pRAR2, E2 responsiveness was observed only in cells cotransfected with wild-type ER or 11C-ER containing a deletion of the DNA-binding domain but not with ER variants that express activation function-1 (AF-1) or AF-2. Using a similar approach, it was shown that the GC-rich sites in RAR1 were also sufficient for ER activation. These results demonstrate that interaction of a transcriptionally active ER/Sp1 complex with GC-rich motifs is required for hormone inducibility of the downstream region of the RAR alpha 1 gene promoter.