The in vitro effects of porcine, salmon and human calcitonin on the K+-evoked overflow of [Met5]enkephalin, substance P and [3H]5-HT (previously taken up) were investigated in superfusion experiments with spinal cord slices. Porcine and salmon calcitonin did not affect the release of [Met5]enkephalin and substance P but enhanced that of [3H]5-HT. In contrast, human calcitonin was inactive. The stimulatory effect of porcine and salmon calcitonin on K+-evoked [3H]5-HT overflow was found with slices from the dorsal or the ventral half of the lumbar enlargement but not with hippocampal or hypothalamic slices. The calcitonin effect on [3H]5-HT outflow persisted in the absence of extracellular Ca2+ but was totally suppressed by 5-HT uptake inhibitors such as citalopram and chlorimipramine and by the 5-HT-releasing agent, p-chloroamphetamine. Direct investigation of the possible action of porcine calcitonin on [3H]5-HT uptake and release demonstrated that the enhanced [3H]5-HT overflow resulted from a p-chloramphetamine-like 5-HT-releasing effect of the hormone at the spinal level. This action might be involved in the potent analgesic effect of intrathecal calcitonin.