External lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contribute to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of the rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases the rVLM activity through elPBN-rVLM projections. Compared to controls, rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P<0.05). Majority of these elPBN neurons contain vesicular glutamate transporter 3. In felines, epicardial bradykinin (BK) and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was reversed (n=6, P<0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn; 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced the rVLM activity evoked by both BK and electrical stimulation (n=5, P<0.05). Furthermore, excitation of elPBN with microinjection of dl-homocysteic acid (DLH; 2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. In contrast, the DLH-enhanced rVLM activity was reversed following iontophoresis of Kyn into the rVLM (n=7, P<0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through monosynaptic excitatory elPBN-rVLM pathways and glutamate mechanism important in elPBN processing of cardiac-cardiovascular sympathoexcitatory responses.