A direct infusion of amphetamine into the dorsal raphe nucleus of the rat inhibited the activity of serotonergic neurons in this site. An intravenous injection of 5-methoxy-N,N-dimethyltryptamine, a serotonin autoreceptor agonist, mimicked this effect. The amphetamine-induced depression of firing rate was blocked by a subsequent injection of methiothepin, a putative serotonin autoreceptor antagonist, but not by pretreatment with a-methyl-p-tyrosine which depletes brain catecholamines. Amphetamine infusions into the surrounding periaqueductal gray or brainstem reticular formation failed to change dorsal raphe activity. The results of these studies indicate that endogenous serotonin, which can be released by a direct infusion of amphetamine, suppresses neuronal activity in the dorsal raphe nucleus by a process of self-inhibition.