The estrogen receptor (ER) is a ligand-activated transcription factor and a member of a large family of nuclear hormone receptors. As a mediator of estrogen hormone action, the ER is involved in many important physiological processes. ER gene expression has been demonstrated to be restricted to certain tissues and under complex hormonal control. However, the molecular mechanisms involved have remained largely unknown. Due to this lack of knowledge an investigation was undertaken to characterize the promoter organization of ER gene and investigate its expression. Approximately 3 kb of the 5' flanking region of the human ER (hER) gene was isolated and sequenced. By performing RT-PCR and RACE experiments it was shown that the hER gene is transcribed from three different promoters. Transcription of the hER gene from these promoters yields three different mRNA isoforms with unique 5' untranslated regions (5'UTRs), but identical coding regions. The expression pattern of the hER mRNA isoforms was investigated by RT-PCR. Both the A- and B-mRNA isoforms were found to be expressed in breast and uterus, whereas expression of the C-transcript was predominantly detected in liver. In bone cells only expression of the B-mRNA could be detected. The steady-state levels of the A- and B-transcripts in normal breast and uterus were quantified and compared with the hER mRNA levels in established cancer cell lines derived from the same tissues. This demonstrated approximately equal levels of the two transcripts in normal tissues whereas the A-mRNA was the most abundant isoform in the cancer cell lines investigated. Approximately 4.5 kb of the 5' flanking region of the rat ER (rER) gene were sequenced. Sequence analysis and PCR experiments suggested that the promoter organization of the rat and human ER genes is only partially conserved which might indicate species-specific differences in the regulation of ER expression. In conclusion, this work suggests tissue-specific alternative promoter usage as a mechanism in the regulation of human and rat ER gene expression.