Puberty involves the maturation of female reproductive circuits to the point of supporting estrogen positive feedback that elicits a luteinizing hormone (LH) surge, triggering ovulation. We have shown that in post-pubertal female rodents, peripheral estradiol (E2) increases progesterone synthesis in hypothalamic astrocytes (neuroP), initiating an LH surge. Interestingly, E2-facilitated neuroP synthesis occurs in adult females but not in males or prepubertal females. The focus of the current study was to determine changes in female astrocytes during puberty that allow E2 to facilitate neuroP synthesis. Of particular interest are responses to estrogen indicated by intracellular calcium flux, levels of estrogen receptor-alpha (ERα)-- trafficked to the cell surface by interaction with caveolin 1 (Cav1) proteins—that couple with metabotropic glutamate receptors (mGluRs) and mediate E2’s induction of neuroP synthesis by activating protein kinase A (PKA). Hypothalamic astrocyte cultures were established from female mice at postnatal day (PND) 23 (prepubertal), PND 35 (pubertal), and PND 60 (adult) and treated with 1nM E2 or vehicle. Calcium imaging revealed that PND 60 astrocytes had a more rapid response to E2 compared to PND 23 astrocytes that had a slower and more sustained response to E2. Surface biotinylation and western immunoblotting determined changes in membrane ERα, Cav1, phosphoPKA. We observed a developmental increase in phosphoPKA and Cav1 proteins (ANOVAs, main effects of age, p values < 0.005; n = 3-11/treatment/age. Surface biotinylation and western blotting revealed an age-related increase in full-length (66 kDa) mERα (ANOVA, main effect of age, p < 0.05, n = 7-11/treatment/age), while there is an age-related decrease in a 36 kDa mERα (ANOVA, main effect of age, p < 0.05, n = 2-6/treatment/age). E2 signaling through the 36 kDa mERα splice variant, which is abundant prior to puberty, could initiate downstream inhibitory signaling cascades attenuating E2-facilitated P4 synthesis. Current studies will determine if the response to E2 in hypothalamic astrocytes undergoes a transition from inhibitory to excitatory signaling due to differential coupling of mERs and mGluRs. These experiments highlight how cellular signaling changes during puberty in hypothalamic astrocytes to allow for neuroP biosynthesis that contributes to estrogen positive feedback. Supported by HD042635 and UL1TR001881.