Antibodies made against serotonin (5HT) were used to identify the serotonin neuronal system in the developing and adult nervous system of Drosophila melanogaster. The 5HT neuronal pattern is composed of a small number of neurons, 84 in larvae and 106 in adults, distributed in clusters composed of one to five neurons in the CNS; 5HT immunoreactive (5HT-IR) neurons appear to be predominantly intrasegmental interneurons; however, intersegmental 5HT-IR fibers are observed and at least some neurons send peripheral fibers. Acquisition of 5HT immunoreactivity in the CNS occurs late in embryogenesis, by 16-18 hours, and most if not all the 5HT neurons appear to persist into adulthood. During early metamorphosis, the intensity of 5HT-IR neuropil transiently decreases. Other changes in the CNS during this period are reflected in the appearance of two new 5HT clusters and 5HT-IR neuropil in the developing optic lobes. Comparison of the 5HT-IR pattern with other transmitter systems in Drosophila as well as comparison of the 5HT-IR pattern within different insect species is presented.