Developmental changes of serotonin-positive fibers with special reference to co-localization with substance P was examined immunohistochemically in the ventral horn of rat lumbar spinal cord. Only about 20% of serotonin-positive fibers co-labelled with substance P on postnatal day (P) 0. The ratio of co-localization gradually increased, and reached the adult value by P28 (60-70%). Enkephalin-positive fibers were not co-localized with serotonin at any age examined. Although the densities of serotonin, substance P and enkephalin per unit area of the ventral horn gradually increased with development up to P28, the density of serotonin in the adult was decreased compared to P28 animals.