We examined whether the altered rostral ventrolateral medulla (RVLM) cholinergic function in spontaneously hypertensive rats (SHR) results from enhanced presynaptic cholinergic tone. Male 12- to 16-week-old SHR and age-matched Wistar Kyoto rats (WKY) were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of cholinergic agents into the RVLM produced a pressor response. The pressor response to physostigmine was greater in SHR than that of WKY whereas the response to ACh and carbachol was the same in WKY and SHR. Bilateral microinjection of scopolamine produced a decrease in blood pressure. The depressor response was greater in SHR than that of WKY. When a microdialysis probe was placed in the RVLM, ACh release in the RVLM was greater in SHR than that of WKY. Choline acetyltransferase (CAT) activity was increased only in the rostro-ventral part of the medulla, which contained the RVLM, but not in other parts of the medulla oblongata. Physostigmine (0.5 mg/kg, i.p.)-induced increases in ACh content were also enhanced only in the rostro-ventral part of the medulla. These results provide direct evidence that ACh release in the RVLM is enhanced in SHR. It appears that the enhanced cholinergic activity in the RVLM of SHR results from an increase in cholinergic impulse flow in the RVLM of SHR. This abnormality may play a role in the maintenance of hypertension in SHR.