The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how the AVPV controls GnRH neurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies <1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating >90% of the evoked fast synaptic currents. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABA(B) receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability.