Recent anatomical studies with horseradish peroxidase injections into the anterior raphe have demonstrated that the nucleus raphe dorsalis in the rat receives a major afferent input from the lateral habenula (LHb). The present study examined electrophysiologically the effects of electrical stimulation of the LHb on the spontaneous activity of midbrain and anterior pontine raphe units in anesthetized rats. The results showed that: (a) LHb stimulation (1 or 10 Hz, 0.5 to 1.0 mA) suppressed the activity of most raphe units, with the effects outlasting the duration of the stimulation in some instances; the raphe cells which showed periods of suppression during LHb stimulation were both those of the classical serotonin type (N = 26), characterized by slow regular baseline firing rates, and other raphe cells (N=52) with faster baseline rates (to 60/s); (b) inhibition of unit activity was much less pronounced for non-raphe cells lateral to the midline; (c) anatomical control stimulation points dorsal to the LHb did not alter raphe unit activity; and (d) the pathway from the habenula to the raphe may involve a dorsal route. After a knife cut through the superior colliculus-central gray at the level of the interpeduncular nucleus, the effects of habenular stimulation were substantially reduced. Conversely, stimulation of the superior colliculus just posterior to the habenula (presumably containing descending fibers from the habenula) markedly suppressed raphe unit activity. In summary, the present electrophysiologic findings were consistent with the view that activation of habenular afferent fibers to the raphe exerted a major inhibitory influence on the spontaneous activity of midbrain and pontine raphe neurons. Considerably smaller effects were exerted on lateral reticular cells. A dorsal pathway may be involved in mediating the habenular effects on raphe activity. © 1979.