Circulating estradiol exerts a profound influence on the activity of the gonadotropin-releasing hormone (GnRH) neuronal network controlling fertility. Using genetic strategies enabling neuron-specific deletion of estrogen receptor α (Esr1), we examine here whether estradiol-modulated GABA and glutamate transmission are critical for the functioning of the GnRH neuron network in the female mouse. UsingVgat- andVglut2-ires-Cre knock-in mice and ESR1 immunohistochemistry, we demonstrate that subpopulations of GABA and glutamate neurons throughout the limbic forebrain express ESR1, with ESR1-GABAergic neurons being more widespread and numerous than ESR1-glutamatergic neurons. We crossedVgat- andVglut2-ires-Cre mice with anEsr1^lox/loxline to generate animals with GABA-neuron-specific or glutamate-neuron-specific deletion ofEsr1. Vgat-ires-Cre;Esr1^lox/loxmice were infertile, with abnormal estrous cycles, and exhibited a complete failure of the estrogen positive feedback mechanism responsible for the preovulatory GnRH surge. However, puberty onset and estrogen negative feedback were normal.Vglut2-ires-Cre;Esr1^lox/loxmice were also infertile but displayed a wider range of deficits, including advanced puberty onset, abnormal negative feedback, and abolished positive feedback. Whereas <25% of preoptic kisspeptin neurons expressed Cre inVgat- andVglut2-ires-Cre lines, ∼70% of arcuate kisspeptin neurons were targeted inVglut2-ires-Cre;Esr1^lox/loxmice, possibly contributing to their advanced puberty phenotype. These observations show that, unexpectedly, ESR1-GABA neurons are only essential for the positive feedback mechanism. In contrast, we reveal the key importance of ESR1 in glutamatergic neurons for multiple estrogen feedback loops within the GnRH neuronal network required for fertility in the female mouse.SIGNIFICANCE STATEMENTCirculating estradiol acts upon the brain to regulate the functioning of many neuronal networks, including those controlling reproduction. Acting in classic homeostatic negative or positive feedback modes, estradiol variably suppresses or enhances the activity of the gonadotropin-releasing hormone (GnRH) neurons throughout the ovarian cycle. We show here that estrogen receptor α (ESR1) within glutamate (VGLUT2) neurons is essential for both the negative and positive estradiol feedback loops. In contrast, ESR1 in GABA neurons is only required for estradiol positive feedback. These studies emphasize the importance of estradiol-modulated amino-acidergic neurons within the GnRH neuronal network and highlight an unexpected prominent role for ESR1-expressing glutamate neurons in fertility control.