In females, ovarian estradiol modulates kisspeptin (Kiss-1) synthesis to act as an obligatory regulator of downstream gonadotropin release in vivo, via stimulation of GnRH neurons. Changes in the ovarian condition are relayed to the neuroendocrine hypothalamus via two sexually dimorphic Kiss-1 populations, located in the anteroventral periventricular (AVPV) and arcuate nuclei, conveying estradiol-positive and -negative feedback, respectively. To elucidate how differential responsiveness to estradiol is mediated in these populations, we generated two kisspeptin-secreting cell lines from an adult kiss1-green fluorescent protein (GFP) female mouse. These lines recapitulate in vivo responsiveness to estradiol, with KTaV-3 (AVPV) cells demonstrating significantly increased kiss1 expression under high physiological estradiol exposure, whereas KTaR-1 (arcuate) cells exhibit kiss1 suppression after lower estradiol exposure. Baseline expression of estrogen receptor-α (esr1) differs significantly between KTaV-3 and KTaR-1 cells, with KTaR-1 cells demonstrating higher basal expression of esr1. Estradiol stimulation of kiss1 expression in KTaV-3 cells is modulated in a dose-dependent manner up to 25.0 pM, with less responsiveness observed at higher doses (>50.0 pM). In contrast, KTaR-1 kiss1 attenuates at lower estradiol doses (2.0–5.0 pM), returning to baseline levels at 25.0 pM and greater. Furthermore, the expression of the core clock genes bmal1 and per2 show normal rhythms in KTaV-3 cells, regardless of estradiol treatment. Conversely, KTaR-1 antiphasic transcription of bmal1 and per2 is phase delayed by low estradiol treatment. Strikingly, estradiol induces circadian rhythms of kiss1 expression only in KTaV-3 cells. Further exploration into estradiol responsiveness will reveal mechanisms responsible for the differential expression pattern demonstrated in vivo between these cell types.