The involvement of the basolateral amygdala in mediating the inhibition of neonatal vocalisation by substance P (NK(1) receptor) antagonists was examined. These studies determined whether the time course for separation-induced vocalisations in guinea-pig pups coincided with NK(1) receptor internalisation (a marker of substance P release) in the amygdala, and whether vocalisations could be blocked by focal injection of the NK(1) receptor antagonist L-760735 into this brain region. The peak period for neonatal vocalisations occurred 5-10 min following maternal separation. This coincided with the peak increase in the number of cells in the basolateral amygdala exhibiting NK(1) receptor endocytosis, consistent with the proposal that substance P is released in the amygdala as a result of isolation stress. Focal injection of L-760735 (15 nmol per side) but not L-770765 (an analogue of L-760735 which has low NK(1) receptor affinity) into the basolateral amygdala attenuated separation-induced vocalisations. In contrast, injection of L-760735 (15 nmol per side) into the dorsal ventricular nucleus of the thalamus, a region with relatively low density of NK(1) receptors, had no effect on neonatal vocalisations. These findings are consistent with other evidence that the amygdala is one possible site of action for the inhibition of neonatal vocalisations by substance P antagonists.