Previous studies have shown that tryptamine (T) is closely associated with the serotonergic system but have failed to determine whether neurons containing only T exist in the raphe nuclei (RN) and/or if T colocalizes with serotonin (5-HT). The use of rabbit-conjugated T antisera (TAS) and rat-conjugated 5-HT antisera (5-HTAS) in a double-labelling technique has made the study of the colocalization of T and 5-HT in neurons of the rat dorsal RN (DR) possible. Slices taken from the same zone of the DR were treated following four different procedures of double-immunolabelling: DAB coloration for the rabbit TAS and DAB-nickel coloration for the rat 5-HTAS either first or second; or DAB coloration for the 5-HTAS and DAB-nickel coloration for the TAS first or second. Control sections were treated according to a single immunoperoxidase staining in the same zone. The immunolabelled neurons were computed using the Biocom 200 program at the same magnification. The results of this double-immunolabelling show that three different cell types exist in the rat DR: (1) T-only-containing neurons, (2) 5-HT-only-containing neurons displaying either homogeneous DAB or homogeneous DAB-nickel, and (3) neurons where T colocalizes with 5-HT (T-positive/5-HT-positive neurons) displaying heterogeneous DAB-nickel coloration. The results were identical whatever the procedure performed or the order of the DAB-nickel revelation.(ABSTRACT TRUNCATED AT 250 WORDS)