The serotonin transporter is an important regulator of serotonergic signaling. In order to analyze where the Drosophila melanogaster ortholog of the mammalian serotonin transporter (dSERT) is expressed in the nervous system, a dSERT antibody serum was used. Ectopic expression studies and loss of function analysis revealed that the dSERT antibody serum specifically recognizes dSERT. It was shown that in the embryonic nervous system dSERT is expressed in a subset of Engrailed-positive neurons. In the larval brain, dSERT is exclusively expressed in serotonergic neurons, all of which express dSERT. dSERT-positive neurons surround almost all brain neuropiles. In the mushroom body of the adult brain, extrinsic serotonergic neurons expressing dSERT engulf the mushroom body lobes. These neurons show regional differences in dSERT and serotonin expression. At the presynaptic terminals, serotonin release is sterically linked to serotonin reuptake. In contrast to this, there are other areas in serotonergic neurons where dSERT expression and/or function are uncoupled from synaptic neurotransmitter recycling and serotonin release. The localization pattern of dSERT can be employed to further understanding and analysis of serotonergic networks.