The preovulatory GnRH/LH surge depends on the presence of estradiol (E(2)) and is gated by a circadian oscillator in the suprachiasmatic nucleus (SCN) that causes the surge to occur within a specific temporal window. Although the mechanisms by which the clock times the LH surge are unclear, evidence suggests that the SCN is linked to GnRH neurons through a multisynaptic pathway that includes neurons in the anteroventral periventricular nucleus (AVPV). Recently, Kiss1 neurons in the AVPV have been implicated in the surge mechanism, suggesting that they may integrate circadian and E(2) signals to generate the LH surge. We tested whether Kiss1 neurons display circadian patterns of regulation in synchrony with the temporal pattern of LH secretion. Mice housed in 14 h light, 10 h dark were ovariectomized, given E(2) capsules (or nothing), and transferred into constant darkness. Two days later, the mice were killed at various times of day and their LH and Kiss1 levels assessed. In E(2)-treated females, LH levels were low except during late subjective day (indicative of an LH surge). Similarly, AVPV Kiss1 expression and c-fos coexpression in Kiss1 neurons showed circadian patterns that peaked coincident with LH. These temporal changes in Kiss1 neurons occurred under steady-state E(2) and constant environmental conditions, suggesting that Kiss1 neurons are regulated by circadian signals. In the absence of E(2), animals displayed no circadian pattern in LH secretion or Kiss1 expression. Collectively, these findings suggest that the LH surge is controlled by AVPV Kiss1 neurons whose activity is gated by SCN signals in an E(2)-dependent manner.