The distribution, morphology and number of serotonin-, catecholamine- and substance P-containing neurons in the human dorsal raphe nucleus were studied. Parallel series of sections were prepared from 10 human brainstems obtained at autopsy from patients without neurological disease aged between 42 and 88 years. The neurons were identified using immunohistochemistry with antibodies raised against phenylalanine hydroxylase (tryptophan hydroxylase-containing, serotonin neurons), tyrosine hydroxylase (catecholamine neurons) and substance P. A reference series of Nissl-stained sections was also prepared and data published separately were used to delineate the subnuclear divisions of the dorsal raphe nucleus and to establish the total number of neurons in each subnucleus. The following principal findings emerged. (1) Serotonin-synthesizing neurons are present in all regions of the dorsal raphe nucleus and their total number is 165,000 +/- 34,000. The same types of neurons as those seen in Nissl material characterize each of the five subnuclei (caudal, dorsal, ventral, ventrolateral and interfascicular). (2) Substance P-containing neurons mostly occupy the rostral part of the nucleus and their number is 74,600 +/- 17,600. (3) Catecholamine cells are only found in the rostral part of the dorsal raphe nucleus and their number is 5600 +/- 3400. (4) In the ventral and interfascicular subnuclei the combined number of serotonin-synthesizing and substance P-containing neurons exceeds the total number of Nissl-stained neurons suggesting that serotonin and substance P co-exist in a substantial part of the cell population of the dorsal raphe nucleus. This is further supported by the highly similar morphology and size of these neurons. It is concluded that there are demonstrable chemical differences between the various subregions of the human dorsal raphe nucleus. These differences are in harmony with the results of hodological studies in animals, which have demonstrated differential projection pathways emerging from this nucleus.