The selective serotonin reuptake inhibitors and tricyclic antidepressants act by inhibiting pre-synaptic reuptake of serotonin (5-HT) leading to elevated synaptic 5-HT concentrations. However, despite extensive efforts little is known about the protein-ligand interactions of serotonin transporter (SERT) and inhibitors. To identify domains and individual amino acids important for ligand binding, we cloned the serotonin transporter from zebrafish, Danio rerio, (drSERT) and compared its pharmacological profile to that of the human serotonin transporter (hSERT) with respect to inhibition of [3H]5-HT uptake and [3H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity towards compounds of the imipramine class, desipramine in particular, exhibiting a 35-fold increased affinity compared to hSERT. drSERT has a 15–30-fold lower affinity towards cocaine and cocaine analogues. The differences in ligand recognition are shown to be primarily caused by interspecies differences in TM10 and were tracked down to three residues (Ala505, Leu506 and Ile507).