This investigation was designed to demonstrate the presence of cholinergic nerve terminals in the pressor area of the ventrolateral medulla (VLPA) and to study the effects of the release of endogenous acetylcholine in this area. Bilateral microinjections (0.1-2 nmol)/site) of 3,4-diaminopyridine (DAP), which releases acetylcholine from cholinergic nerve terminals, into the VLPA in anesthetized rats evoked an increase in blood pressure and heart rate which lasted for 20-40 min. Intravenous injections of the same doses of this agent failed to evoke a response. The ganglion blocker, chlorisondamine (3 mg/kg, i.v.) abolished the responses to microinjections of DAP indicating that the responses were mediated by the sympathetic nervous system. Microinjections of scopolamine or a specific M2 muscarinic receptor antagonist (AFDX-116) into the VLPA prevented the pressor and tachycardic responses to subsequent microinjections of DAP at the same sites indicating that the responses were mediated via M2 receptors. Microinjections of hemicholinium (3 nmol/site; which impairs acetylcholine synthesis) attenuated the responses to the subsequent microinjections of DAP at the same sites. These results indicate that the substance released from the terminals in the VLPA may be predominantly acetylcholine which evokes pressor and tachycardic responses via M2 muscarinic receptors. The origin and physiological significance of these cholinergic terminals in the VLPA are not known.