Substance P (SP) is a neuropeptide contained in axon terminals. Various classical neurotransmitters coexist with SP in mammalian brains, but there has been no information on the colocalizing substances in the central nucleus of amygdala (CeA), where both SP and its specific receptor are highly concentrated. The present study aimed at determining the colocalizing neurotransmitter in SP terminals in CeA by multi-label immunohistochemistry combined with digitized quantitative analysis. Unexpectedly, most of SP-containing boutons did not show immunoreactivities for any of the transmitters or their marker proteins examined (GABA, glycine, glutamate, acetylcholine, serotonin, or dopamine). Electron microscopy demonstrated small clear vesicles in addition to dense core vesicles within SP-positive terminals that formed symmetrical synapses, indicating the presence of some classical neurotransmitter, most likely GABA. Therefore tissues were fixed by zinc-aldehyde to enhance immunoreactivity for a low level of glutamic acid decarboxylase (GAD), the GABA synthetic enzyme. This led to weak but consistent labeling for GAD in the majority of SP-positive boutons in CeA. By contrast, definite GAD-immunoreactivity was confirmed in SP-containing boutons in the substantia nigra pars reticulata even in specimens treated with a conventional fixative, indicating that negligible GAD labeling in CeA is not ascribed to methodological problems such as interference by the presence of SP but actually reflects low GAD content. These data suggest a unique mode of synaptic transmission at amygdalar SP-containing terminals where slowly-acting SP is concentrated but both GABA and its synthetic enzyme are maintained at low levels, possibly underlying long-lasting responses in emotions.