We examined whether baroreceptor activation causes a release of acetylcholine (ACh) in the rostral ventrolateral medulla (RVLM) of the rat, in order to investigate a possible connection between RVLM cholinergic systems and cardiovascular baroreflexes. Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Either electrical stimulation of aortic nerve or baroreceptor activation by intravenous phenylephrine produced an increase of the release of ACh in the RVLM, whereas baroreceptor denervation and tetrodotoxin (TTX) microinfusion in the RVLM inhibited the increase in ACh release induced by phenylephrine. TTX injected in the caudal ventrolateral medulla (CVLM) inhibited the phenylephrine-induced increase of ACh release. The excitatory amino acid L-glutamate microinfused in the CVLM produced an release in ACh release in the RVLM. These results suggest that there is a connection between RVLM cholinergic systems and cardiovascular baroreflexes. It is probable that neurons in the CVLM are involved in mediating the release of ACh in the RVLM.