The purpose of this study was to identify factors from astrocytes that can regulate LHRH neurosecretion. Exposure of LHRH-secreting (GT1–7) cells to conditioned media (CM) from C6 glial cells and hypothalamic astrocytes (HA) stimulated LHRH release. Assays of C6 and HA CM revealed that transforming growth factor-β₁ (TGF-β₁) and 3α-hydroxy-5α-pregnane-20-one (3α,5α-THP), both known LHRH secretagogues, were present in CM and their levels increased in parallel to the LHRH-releasing activity of CM. In contrast, TGF-α was undetectable in C6 or HA CM. Ultrafiltration to remove peptides with molecular weights >10 kDa virtually abolished the LHRH-releasing ability of the HA CM. Furthermore, immunoneutralization with a panspecific THF-β antibody dose-dependently attenuated the LHRH-releasing activity of the CM. Rat hypothalamus and GT1–7 cells were demonstrated to express TGF-β receptors as well as furin, an enzyme that converts latent TGF-β₁ to active TGF-β₁. Estrogen receptor-α and ER-β mRNA and protein were also demonstrated in HAs by reverse transcription-polymerase chain reaction and double immunofluorescence, and treatment with 17β-estradiol (17β-E₂) increased both active and latent TGF-β₁ levels in HA CM. The effect of 17β-E₂ was completely blocked by the ER antagonist ICI8280. As a whole, these studies provide evidence of a previously undescribed 17β-E₂-TGF-β₁-LHRH signaling pathway.