The dorsal raphe nucleus (DR) of the rat was studied with triple labeling immunofluorescence histochemistry to evaluate the quantitative relationships between neurons expressing 5-hydroxytryptamine (5-HT), the neuropeptide galanin (GAL) and the nitric oxide (NO) synthesizing enzyme NO synthase (NOS). In addition retrograde tracing studies were performed. It could be established that a high percentage (between 40 and 60%) of the 5-HT neuron profiles in the ventromedial and dorsomedial subgroups of the DR contained both GAL and NOS after colchicine treatment. This triple coexistence was lower in the dorso-lateral subgroup and much lower in the lateral subgroup (down to 5%). All GAL neuron profiles contained 5-HT, and they constituted up to 80% of all labeled profiles in the rostral ventromedial and caudal dorsomedial subgroups, with the lowest percentage in the lateral subgroup (45%). The percentage of 5-HT-alone neuron profiles in these four subgroups varied between 15-40%. The proportion of 5-HT/NOS neurons was low (a few percent) at all levels, and this was the case also for NOS-alone neuron profiles except in the lateral subgroup, where 10-20% were of this type. It could be established that some 5-HT/GAL/NOS neurons project to the striatum. These retrogradely labeled cells were mainly found in the mid-line subgroups. In the striatum a moderately dense 5-HT fiber network and numerous NOS-positive cell bodies and fibers could be observed. However, only a few, weakly fluorescent GAL fibers were found and in a small number of cases it could be shown or was likely that 5-HT and GAL coexisted. No evidence for coexistence between 5-HT and NOS was obtained. The present findings strongly suggest that a large proportion of the DR 5-HT neurons can synthesize and release two additional messenger molecules, GAL and NO. Furthermore, even if the 5-HT/GAL/NOS neurons project to the striatum, the amounts of GAL and NOS transported to the terminal ramifications in this area are very low. This is in agreement with a very low GAL synthesis in the DR under normal circumstances, which is also indicated by the fact that colchicine treatment is needed to visualize GAL-like immunoreactivity in DR cell bodies. It is possible that NO, and perhaps GAL, in 5-HT neurons exert their main actions at the somatic and dendritic level in the DR.