Afferent connections of the serotonin (5-HT)-containing dorsal raphe nucleus were investigated in the rat utilizing the horseradish peroxidase (HRP) retrograde cell labeling technique. Small quantities (0.1-0.5 mul) of HRP solutions were infused into the dorsal raphe, and the brains were examined 19-72 h later for retrograde transport of the enzyme. Intrinsic connections within the dorsal raphe nucleus were revealed by this mapping technique, as was an input to the dorsal raphe from another serotonergic cell group, the median raphe nucleus. Little evidence was found for projections from other, more remote, brain sites. A serotonergic innervation of the dorsal raphe was also demonstrated by the presence of high affinity uptake of [3H]5-HT (Km=0.17 muM) into synaptosomal suspensions of the dorsal raphe nucleus. Synaptosomal uptake of [3H]5-HT was blocked by selective destruction of serotonergic axon terminals induced by the intraventricular injection of 200 mug of 5,7-dihydroxytryptamine following desipramine HCl pretreatment, but not by destruction of catecholaminergic axon terminals induced by intraventricularly injected 6-hydroxydopamine (2 X 250 mug). The uptake of [3H]-5-HT by synaptosomes of the dorsal raphe was comparable to that of striatal and hypothalamic synaptosomes, and markedly greater than that of synaptosomes from the cerebellum or nearby dorsal central gray or midbrain reticular formation, indicating the presence of a relatively dense serotonergic innervation. These data together indicate that neurons in the dorsal raphe nucleus receive a prominent serotonergic input that is derived, at least in part, from other neurons within the dorsal nucleus and from a neighboring raphe nucleus.