Two domains of the human estrogen receptor, responsible for hormone binding (region E) and tight nuclear binding (region C), are essential for the receptor to activate efficiently the transcription of estrogen-responsive genes. Region D, which joins the DNA- and hormone-binding domains, can be altered without affecting activation. Deletion of the N-terminal domain (region A/B) has no effect on activation of a reporter gene containing a vitellogenin estrogen-responsive element (ERE) and the HSV-tk promoter, whereas it severely impairs activation of the human pS2 gene promoter. Deletion of most or all of the hormone-binding domain leads to only about 5% constitutive transcriptional activity, yet these mutants appear to bind efficiently to an ERE in vivo. Apparently, region C recognizes the ERE of target genes, and the hormone-binding domain plays an essential role for efficient activation of transcription.