A cocaine-sensitive, high-affinity Drosophila serotonin (5-hydroxytryptamine; 5HT) transporter cDNA, denoted dSERT1, was isolated and characterized in oocytes. dSERT1 shows little transport of other monoamines and is Na+ and Cl- dependent. Sequence analysis indicates 12 putative transmembrane domains and strong homologies (approximately 50%) among dSERT1 and mammalian 5HT, norepinephrine, and dopamine transporters. Interestingly, the pharmacological properties of dSERT1, including sensitivity to antidepressants, are more similar to those of mammalian catecholamine transporters than to mammalian 5HT transporters. Two-electrode voltage-clamp analysis demonstrated 5HT-induced, voltage-dependent currents. Cloning and characterization of dSERT1 adds significantly to our knowledge of the diversity of 5HT transporters with regard to primary sequence, pharmacological profile, and permeation properties.