Because of the biochemical colocalization of the 5-HT3 receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT3 receptor function and its modulation by antidepressants. Treatment with methyl-β-cyclodextrine (MβCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MβCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT3 receptor were retained. Although both the 5-HT3 receptor and flotillin-1 were predominantly found in raft-like domains in Western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT3 receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT3 receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT3 receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel.