Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species-specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species-specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO-mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species-specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.