We previously demonstrated that tryptophan hydroxylase (TPH), the rate-limiting enzyme of serotonin (5-HT) synthesis, was commonly present in the brains of some insects. The current study was aimed at determining the number of serotonergic neurons in the brain and suboesophageal ganglion of adult Drosophila melanogaster and to investigate further the differences in immunoreactivity between 5-HT and TPH. Brain sections of Drosophila were immunostaind with sheep anti-TPH polyclonal antibody and rabbit anti-5-HT antiserum. The 5-HT-like immunoreactive neurons were also immunoreactive for TPH and bilaterally symmetrical; 83 neurons were found in each hemisphere of the brain and suboesophageal ganglion of adult Drosophila. This technique of colocalizing 5-HT and TPH revealed a larger number of serotonergic neurons in the brain and suboesophageal ganglion than that previous reported, thus updating our knowledge of the 5-HT neuronal system of Drosophila.