Several studies have shown that neuropeptide Y (NPY) is involved in the stimulation of gonadotropin hormone releasing hormone (GnRH) and luteinizing hormone (LH) secretion and that these effects are modulated by gonadal steroid feedback. The NPY regulation of GnRH release is probably mediated by the activation of the Y<sub>1</sub> receptor subtype. In this study we examined the regulation of the Y<sub>1</sub> receptor gene transcription by estrogens in transiently transfected NG108-15 neuroblastoma glioma cells. A chimeric plasmid containing the murine Y<sub>1</sub> receptor promoter fused to the firefly luciferase reporter gene was induced by approximately 2-fold in response to 17β-estradiol treatment. The estrogen-mediated enhancement of luciferase activity was dose-dependent, blocked by the estrogen receptor (ER) antagonist ICI 182,780, and was strictly dependent on the presence of ERα, since it occurred only in NG108-15 cells cotransfected with an expression vector for the human ER. Mutational analysis was performed to investigate whether the hemipalindromic estrogen-responsive elements (EREs) flanking the Y<sub>1</sub> receptor gene are responsible for conferring estradiol inducibility to the Y<sub>1</sub> receptor gene promoter. Mutation of the ERE1 half site at position –932, or mutation of the ERE2 half site at position –809, relative to the ATG, failed to affect the 17β-estradiol-mediated enhancement of luciferase activity. Conversely, mutation of both ERE1 and ERE2 half sites completely abolished activation of luciferase activity induced by estrogen. We also examined whether 17β-estradiol stimulates the transcriptional activity of the Y<sub>1</sub> receptor gene by binding to ERβ. Results demonstrated that luciferase activity was not modulated by estrogens when cells were transfected with the expression plasmid bearing the human ERβ. Moreover coexpression of both ERα and ERβ completely abolished the estrogen-induced activation of luciferase activity observed in the presence of ERα. Our data suggest that estrogens activate Y<sub>1</sub> receptor gene transcription possibly via a direct interaction of ERα with the hemipalindromic EREs flanking the Y<sub>1</sub> receptor gene.