The exact molecular mechanisms regulating estrogen receptor (ER)alpha expression in breast tumors are unclear, but studies suggest that the regulation is at least partly transcriptional. We therefore undertook a detailed analysis of ERalpha promoter activity in a number of breast cancer cell lines. We find that the majority of ERalpha promoter activity lies within the first 245bp of the 5'-flanking region of the gene. Three elements essential for full ERalpha promoter transcriptional activity were identified within the -245 to -192bp region in transient transactivation assays using linker-scanner mutation analysis. These three elements include two binding sites for the Sp1 family of transcription factors as well as a non-consensus E box. We show that both Sp1 and Sp3 bind to this region using electrophoretic mobility shift assays. Exogenous expression of Sp1 or Sp3 in Sp1/3-negative Drosophila Schneider SL2 cells results in transactivation of the -245 to +212bp fragment of the ERalpha promoter. These data demonstrate that transcription of ERalpha is dependent upon the expression of members of the Sp1 family.