AbstractIn the dorsal raphe nucleus, 17β‐estradiol (E2) increases the expression of the brain‐specific, rate‐limiting enzyme for serotonin biosynthesis, tryptophan hydroxylase‐2 (Tph2). Although estrogen receptor beta (ERβ) has been localized to Tph2 neurons, little is known about the transcriptional regulation of the Tph2 gene by estrogen. Since the ERβ agonist, diarylpropionitrile (DPN) also increases Tph2 expression, we tested the hypothesis that E2 regulates the Tph2 promoter through direct interactions with ERβ. A serotonergic cell line, B14, which endogenously expresses ERβ was transiently transfected with a fragment of the human TPH2 5′‐untranslated region (5′‐UTR) cloned into a luciferase reporter vector (TPH2‐luc). Treatment with E2 or DPN caused a dose‐dependent increase of TPH2‐luc activity. In contrast, E2 conjugated to bovine serum albumin, which is cell membrane impermeable, had no effect on TPH2‐luc activity. An estrogen receptor (ER) antagonist blocked E2 or DPN‐induced TPH2‐luc activity suggesting a classical ER mechanism. In silico analysis revealed an estrogen‐response element (ERE) half‐site located within the TPH2 5′‐UTR. Deletion and site‐directed mutation of this site abolished ligand‐induced TPH2‐luc activity. These results support the concept that there is a direct and functional interaction between E2:ERβ and the ERE half‐site of the TPH2 promoter to regulate Tph2 expression. image We illustrate a direct regulation of the TPH2 transcription by estradiol and ERβ via a newly identified ERE half‐site within the TPH2 promoter: (i) Estradiol‐ or an ERβ agonist‐induced TPH2 transcription was blocked by an ER antagonist, while (ii) membrane impermeable form of estradiol did not induce transcription. (iii) Deletion or mutation of the ERE half‐site abolished ligand‐induced TPH2 transcription.