Experiments were performed to determine whether the pathway by which median raphe stimulation increases blood pressore involves serotonergic terminals in the anterior hypothalamic/preoptic area and cholinergic terminals in the posterior hypothalamus. Injection of 5,7-dihydroxytryptamine into the median raphe nucleus decreases serotonin and turnover of acetylcholine in the hypothalamus of the rat. The hypertensive action of injection of serotonin into the anterior hypothalamic/preoptic area is blocked by injection of atropine or hemicholinium-3 into the posterior hypothalamus and by i.c.v. injection of hemicholinium-3 when measured in urethane-anesthetized rats. However, the pressor effect of electrical stimulation of the median raphe nucleus is not blocked by the intrahypothalamic injection of atropine or hemicholinium-3, only partially blocked by i.c.v. hemicholinium-3 and not blocked by specific lesioning with 5,7-dihydroxytryptamine. Moreover, injection of metergoline into the anterior hypothalamic/preoptic area blocks the pressur response to hypothalamic serotonin but does not block the pressor response to median raphe stimulation. Stimulation of the median raphe nucleus does not affect acetylcholine turnover in the hypothalamus. These results suggest that serotonergic mechanisms in the anterior hypothalamic/preoptic area exert their pressor effect through a pathway with a cholinergic link in the posterior hypothalamus. Serotonergic neurons arising in the median raphe nucleus probably are not involved in this pathway through the hypothalamus, although a cholinergic pathway may be involved elsewhere in their pressor actions. The effect of median raphe nucleus lesion on turnover of acetylcholine in the hypothalamus is most likely unrelated to regulation of blood pressure.