Acetylcholine in the central nervous system appears to be involved in some aspects of hypertension. Clonidine and methyldopa may inhibit acetylcholine (Ach) release in several brain areas. The present study was therefore designed to determine whether a depletion of brain Ach could modify the antihypertensive effect of clonidine in freely moving spontaneously hypertensive (SHR), Grollman hypertensive (GHR) and DOCA-salt hypertensive (DHR) rats. Intravenous injection of clonidine (15, 30 and 75 micrograms/kg) reduced mean arterial pressure (MAP) and heart rate (HR) in all hypertensive animals. The hypotensive effect was more marked in SHR than in DHR and GHR. The effect was also reproducible when the drug dose was repeated 3 h later in rats pretreated with saline (5 microliters) in the lateral cerebral ventricle (i.c.v.). When clonidine administration was repeated in hypertensive animals 3 h after i.c.v. hemicholinium-3 (20 micrograms/5 microliters), the decrease in MAP and HR was significantly reduced compared with that observed in the same animals after the first injection. The data suggest that the antihypertensive effect of clonidine depends partially upon the integrity of central cholinergic neurons.