We have recently isolated two untranslated first exons, exon ON and exon OS, of rat estrogen receptor alpha (ERα) gene from the liver by use of 5' -rapid amplification of the cDNA ends (5'-RACE) method. In this communication, we further analyzed the 5'-untranslated region (UTR) of the ERα mRNA in rat anterior hypophysis in order to investigate the existence of the other 5'-untranslated exon(s) of rat ERα gene. Total RNA from the anterior hypophysis of 8-week-old female Wistar strain male rats was subjected to 5' -RACE with antisense primers located in exon 1 of the rat ERα gene and one of the positive clones (clone 35) was sequenced. The nucleotide sequence of clone 35 revealed the insertion of a previously unidentified exon (which we termed “exon 0T”) between exon 0 (the first reported 5'-UTR form of rat ERa mRNA) and exon 1 of rat ERα mRNA. Analysis of rat genomic DNA indicated that exon 0T was located between exon 0 and exon 1 of rat ERα gene. We further investigated the distribution of ERα mRNA containing exon 0T in several brain regions and various peripheral tissues of 8-week-old male and female Wistar strain rats by use of reverse transcription-polymerase chain reaction (RT-PCR). The distribution of the ERα mRNA (0T-1) was essentially similar to that of ERα mRNA in which exon 0 was spliced onto exon 1 reported previously. These results indicate that (1) exon 0T is a novel untranslated exon of rat ERα gene which is located between exon 0 and exon 1 on rat genomic DNA, (2) exon 0T is inserted between exon 0 and exon 1 of ERα mRNA by alternative splicing, and (3) this alternative splicing may occur in tissues where the transcription of ERα gene is initiated from exon 0.