GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH/LH release and GnRH neuron firing activity and may be involved in estradiol feedback, but the neurobiological mechanisms for these actions are unknown. G protein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting both direct and indirect effects are possible. To investigate this and determine whether kisspeptin activation of GnRH neurons is estradiol sensitive, we recorded the firing rate of GnRH neurons in brain slices from adult female mice that were ovariectomized (OVX) and either treated with estradiol (E) capsules (OVX+E) or left without further treatment. Kisspeptin increased GnRH neuronal activity in a dose-dependent manner in cells from both OVX and OVX+E mice, and estradiol significantly potentiated the response. To begin to distinguish direct from indirect actions of kisspeptin, fast synaptic transmission mediated by ionotropic gamma-aminobutyric acid and glutamate receptors was pharmacologically blocked (blockade). Blockade reduced GnRH response to kisspeptin in OVX+E but not in OVX mice. Actions of kisspeptin were also assessed using whole-cell voltage- and current-clamp recording in slices from OVX animals. Kisspeptin application depolarized GnRH neurons in current-clamp and generated inward current in voltage-clamp recordings, even after blocking action potential-dependent neural communication, consistent with a direct effect. Blockers of potassium channels abolished the inward current. Together our data indicate that kisspeptin activates GnRH neurons via both direct and transsynaptic mechanisms and that transsynaptic mechanisms are either enabled and/or potentiated by estradiol.