The 5-hydroxytryptamine-3 (5-HT3) receptor, a ligand-gated ion channel, is known to be involved in gut motility and peristalsis, the mediation of pain and psychiatric diseases. 5-HT3 receptor antagonists are effectively used to treat chemotherapy-induced emesis and irritable bowel syndrome. We have characterized the impact of four naturally occurring variants in the HTR3B gene leading to amino acid exchanges within the respective subunit of heteromeric 5-HT3A/B receptors on a functional and expressional level.For functional characterization, a Ca influx assay based on aequorin bioluminescence was used. Radioligand-binding studies with the 5-HT3 receptor antagonist [H]GR65630 were carried out to determine expression levels of heteromeric 5-HT3A/B receptors. Transiently transfected human embryonic kidney 293 cells using 5-HT3A and 5-HT3B complementary DNA constructs were shown to coexpress homopentameric 5-HT3A next to heteromeric 5-HT3A/B receptors. The variant p.V183I decreased surface expression, whereas p.Y129S and p.S156R led to pronounced increases of 5-HT maximum responses, despite nearly unaltered surface expression levels of heteromeric 5-HT3A/B receptors.These results may help to explain earlier reported association findings of the frequent p.Y129S and p.V183I variants with psychiatric diseases. Replication studies with larger sample pools, especially regarding the rare p.S156R variant would be useful, to obtain an idea about the predisposing role of these single nucleotide polymorphisms as susceptibility variants.