The ventral medulla oblongata of rats was analyzed with a double-labelling immunofluorescence technique using guinea pig antibodies directed against serotonin (5-HT) and rabbit antisera directed against enkephalin (ENK). Numerous cells in the region of nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus showed immunostaining for either 5-HT or ENK. A substantial number of cells showed positive immunostaining for both 5-HT and ENK. 5-HT/ENK double-labelled cells were most frequently encountered in an area that extended from the rostral aspect of the inferior olivary nucleus to the pontomedullary border. This region corresponds anatomically to nucleus raphe magnus/nucleus paragigantocellularis. In addition, a number of the 5-HT/ENK-containing cells were retrogradely labelled with Fluoro-Gold dye that had been injected into the thoracic spinal cord several days prior to perfusion. Schematic drawings showing the anatomical distribution of 5-HT/ENK colocalization are provided.