The central nucleus amygdala (CeA), a limbic forebrain structure, has an important role in the integration of emotional and cardiovascular responses (Iwata et al. 1987; Dampney, 1994). We have earlier confirmed that there is a projection from the CeA that forms GABAergic synapses with neurons in the nucleus of the solitary tract (Saha et al. 2000), the medullary site of the first synapse for afferent fibers from baroreceptors, chemoreceptors and cardiac receptors, and have recently demonstrated that CeA neurons project directly to blood pressure regulating neurons in the rostral ventrolateral medulla (RVLM, Saha et al. 2005). The RVLM contains sympathetic premotor neurones and plays an important role in blood pressure regulation through its projections to preganglionic sympathetic neurons in the spinal cord. The present study was designed to investigate the neurochemical nature of CeA terminals in the RVLM by using anterograde tracing with vesicular glutamate transporter (VGLUT1, VGLUT2) immunocytochemistry. All experiments were performed on 280-300g rats anaesthetised with halothane (5% in O₂). The CeA terminals in the medulla were labelled by microinjecting an anterograde tracer, biotin dextran amine (BDA, 0.2 μl of 10% in saline) stereotaxically into the CeA. Following 7-10 days survival time, with buprenorphine analgesia as required, the rats were re-anaesthetised and perfused with 4% paraformaldehyde ± 0.05% glutaraldehyde fixative, and vibratome sections of the brain stem were processed for confocal and electron microscopy. For confocal microscopy, BDA-labelled terminals were visualised with streptavidin Alexa488 (green fluorescence) and VGLUTs were visualised by incubating sections in antibodies to VGLUT1 or VGLUT2 raised in rabbit (gift of Dr J D Erickson; Varoqui et al. 2002) and then in anti-rabbit IgG conjugated to Cy3 (red fluorescence). For electron microscopy, BDA-labelled terminals were visualised by incubating sections in avidin-biotin complex and reacting with diaminobenzidine and hydrogen peroxide. The glutamate transporters were visualised with 1nm gold particles conjugated to second antibodies. Injections of BDA into the CeA resulted in anterogradely labelled axons and axon terminals in the RVLM. At the confocal microscopic level, CeA terminals in the RVLM contained VGLUT2 but not VGLUT1 immunoreactivity. At the electron microscopic level, many of the axon terminals were found to make asymmetric synapses and contain VGLUT2 immunoreactivity. The results suggest that neural projections from the CeA to the RVLM are predominantly excitatory in nature. The CeA may influence the sympathetic outflow during stressful situation via these direct excitatory projections to RVLM neurons.