Published data on the rat estrogen receptor (rER) gene have so far provided evidence for only one promoter. In contrast, the human ER (hER) gene has been reported to be transcribed from three different promoters A, B and C. In order to facilitate a more in-depth analysis of the promoter organization in the rER gene 4.5 kb of its 5' flanking region were isolated. Sequence analysis suggests that the rER gene is transcribed from a promoter homologous to promoter B in the hER gene. However, the region corresponding to the human promoter A is much more divergent between the species and rapid amplification of cDNA ends (RACE)-PCR as well as RT-PCR experiments support the notion that the rER gene does not contain a functional A promoter. Interestingly, a novel rER mRNA isoform containing a previously unknown 5' untranslated exon was isolated by RACE-PCR, suggesting the existence of a second, distal promoter in the rER gene. Our results show that this ER mRNA isoform C is the only one expressed in rat liver. It thus appears that the rER gene contains two promoters that are utilized in a tissue-specific fashion.