Kisspeptin is a critical regulator of the hypothalamic-pituitary-gonadal axis, via its action on gonadotropin releasing hormone (GnRH) neurons. In rodents, the preovulatory activation of GnRH neurons is under the control of the suprachiasmatic nucleus (SCN), the central biological clock. Kisspeptin-synthesizing neurons located in the rostral periventricular area of the third ventricle of the hypothalamus (RP3V) are thought to integrate these circadian signals with estrogen (E₂) feedback to appropriately time the preovulatory GnRH surge. Consistent with this, kiss1 gene expression in the RP3V as well as the luteinizing hormone surge are controlled by the vasopressin (AVP)-rich dorsomedial SCN. Furthermore, RP3V kisspeptin neurons are innervated by AVP-containing fibers originating in the SCN, suggesting that AVP might play a key role in conveying circadian cues to RP3V kisspeptin neurons. Here, we investigated the effect of exogenous AVP on the electrical activity of RP3V kisspeptin neurons in acute brain slices from kisspeptin-IRES-Cre x R26 tGFP female mice. Our results show that bath application of AVP causes a significant and reversible increase in the spontaneous firing in a majority (>60 %) of RP3V kisspeptin neurons from intact mice. This effect does not require fast synaptic transmission, and involves the activation of type I AVP receptors. AVP-induced increase in RP3V kisspeptin neuron firing is similar in slices from diestrous and proestrous females, but is markedly reduced in slices from ovariectomized (OVX) mice. Further experiments show that the AVP effect on RP3V kisspeptin neurons is restored to ‘ovary-intact’ levels by in vivo treatment of OVX mice with E₂ implants. Together our results provide a cellular mechanism through which E₂ may gate the responsiveness of RP3V kisspeptin neurons to activation by the SCN.