Developmental changes in the coexistence of serotonin and substance P/enkephalin within single fibers of lamina IX of the rat lumbar spinal cord were examined by the use of a double-labelling immunohistochemical technique. On postnatal day (P) 0, 65.0% of immunoreactive varicosities contained only serotonin, and 21.3% of them had both serotonin and substance P. The coexistent ratio of serotonin and substance P in single fibers increased with development: 61.9% of serotonin positive varicosities co-contained substance P on P28, similar to the ratio found in adult animals (67.4%). The ratios of varicosities containing only substance P remained the same from P0 to adult stage (about 15%). Enkephalin positive immunoreactivity was not co-localized with serotonin positive varicosities at any stage of development. Although numerous serotonin positive fibers were found in lamina IX, only a few substance P and enkephalin positive fibers were observed in the same area on P0. The density of serotonin positive varicosities increased slightly by P28, whereas substance P and enkephalin positive fibers increased considerably by this age. Between P28 and the adult stage, the density of serotonin positive fibers decreased by about 50%. The cross sectional area of axonal varicosities containing serotonin- and substance P-like immunoreactivity was similar in both P0 and adult animals, whereas that of enkephalin positive fibers was different. We also examined the coexistence of serotonin and substance P within single neurons of the caudal raphe nuclei in P7 and adult animals, and found that the coexistent ratio significantly increased with development.