We examined whether reticulospinal sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM) have muscarinic receptors and ACh inputs, and whether these cholinergic mechanisms on RVLM neurons are involved in the pressor response induced by peripheral administration of physostigmine. Microiontophoretic application of ACh and carbachol enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of RVLM neurons by these cholinoceptor agonists was abolished by the nonselective muscarinic receptor antagonist scopolamine and/or by the M2 muscarinic receptor antagonist methoctramine. Physostigmine and the ACh releaser 3,4-diaminopyridine also enhanced the firing rate of RVLM neurons. Intravenous administration of physostigmine enhanced RVLM sympathoexcitatory neuronal activity and the physostigmine-induced response was reversed by iontophoretic application of scopolamine onto the neurons. These results are consistent with the hypothesis that M2 muscarinic receptors responsible for blood pressure regulation are present on RVLM sympathoexcitatory neurons and these receptors receive ACh inputs. Physostigmine injected systemically may exert a portion of its hypertensive effect through a direct enhancement of cholinergic mechanisms on RVLM sympathoexcitatory neurons.