Objective: We demonstrated previously that central muscarinic cholinergic receptor (mAChR) activation increased splanchnic sympathetic nerve activity and sympathetic baroreflex function via activation of mAChR in the rostral ventrolateral medulla (RVLM), and we found that some RVLM bulbospinal neurons contain muscarinic M2R mRNA. Here, we examined the gene expression, cellular distribution and functional role of muscarinic receptors in the RVLM in spontaneously hypertensive rats (SHR) compared with Wistar–Kyoto (WKY) rats. Method and results: Using the sensitive technique of quantitative real time reverse transcriptase-PCR, M2R mRNA level was elevated two-fold (P < 0.05) and M4R mRNA was downregulated two-fold (P < 0.001), with all other receptors expressed at similar levels, in the rostral ventral medulla of SHR compared with WKY. Bulbospinal, but not catecholaminergic neurons, in the RVLM expressed M2R mRNA (M2R+), and similar numbers were found in the RVLM of SHR and WKY. Could elevated M2R within individual neurons or enhanced presynaptic activity reflects enhanced cholinergic effects in the RVLM? Activation of central mAChR using oxotremorine evoked a larger increase in mean arterial pressure in SHR compared with WKY (P < 0.01); however, oxotremorine-induced increases in splanchnic sympathetic nerve activity, and sympathetic baroreflex function were similar in SHR and WKY. Conclusion: These data indicate that enhanced pressor responses in SHR, following centrally mediated mAChR activation, are not associated with RVLM-mediated constriction of the splanchnic circulation or effects on the sympathetic baroreflex, but could reflect modified mAChR gene expression elsewhere. RVLM-dependent splanchnic sympathetic nerve activity effects, evoked by mAChR activation, are not mediated by the differential M2/M4 receptor mRNA levels identified in SHR compared with WKY.