The accessory olfactory system constitutes a sensory system specifically involved in regulating neuroendocrine function and reproductive behavior. The chemosensitive structure of this system, the vomeronasal organ, exclusively innervates the accessory olfactory bulb, which in turn projects via monoor disynaptic pathways to a limited number of regions implicated in endocrine and sexual function, including the amygdala and hypothalamus. The present study investigated synaptic processing between the different levels of this sensory system, with particular focus on the input from the accessory olfactory bulb to the medial amygdala and the reciprocal connections between the medial amygdala and the ventromedial hypothalamus. Extracellular single-unit recordings were obtained from medial amygdala neurons in anesthetized female rats to study the synaptic responses elicited by stimulation of the olfactory bulb and hypothalamus. Locally applied agonists and antagonists of amino acid neurotransmitters were tested for their ability to mimic and block these synaptic responses in an attempt to identify the neurotransmitters involved in the stimulated pathways. Electrical stimulation of the accessory olfactory bulb induced orthodromic responses in 88% of 288 medial amygdala neurons, with 67% showing orthodromic inhibition and 21% showing excitation as the shortest-latency response. Many cells displayed multiphasic responses with both orthodromic excitation and inhibition. In separate experiments, main olfactory bulb stimulation also produced excitatory (39%) or inhibitory (50%) orthodromic responses in medial amygdala neurons (n = 105), but the latency and distribution of responses were significantly different than with accessory olfactory bulb stimulation. Stimulation of the ventromedial hypothalamus also had prominent excitatory (29%) or inhibitory (59%) orthodromic actions on the majority of amygdala neurons. Out of 288 cells tested with both accessory olfactory bulb and ventromedial hypothalamus stimulation, 79% were orthodromically responsive to both areas, whereas only 3% responded to neither area. Fourteen (6%) medial amygdala neurons were antidromically activated from the ventromedial hypothalamus, and 11 of these 14 cells were simultaneously orthodromically responsive to accessory olfactory bulb stimulation, indicating that the accessory olfactory bulb can influence medial amygdala neurons that project directly to the hypothalamus. Iontophoretic application of drugs to the medial amygdala revealed that glutamate and GABA consistently mimicked the orthodromic excitatory and inhibitory responses, respectively, from both the accessory olfactory bulb and ventromedial hypothalamus. In some cases, ejection of bicuculline could block or reverse excitation of the orthodromic inhibitory responses, whereas kynurenic acid could eliminate the orthodromic excitation, indicating that excitatory and inhibitory amino acid transmitters are at least partially involved in these pathways. These findings form a framework for determining the synaptic mechanisms underlying information processing in the accessory olfactory system and olfactory modulation of reproductive function.