Serotonin type 3 (5-hydroxytrptamine-3, 5-HT₃) receptors are ligand-gated cation channels present in both central and peripheral nervous systems. In humans there are five different subunits (A, B, C, D and E) of 5-HT₃ receptors which can form homomeric or heteromeric receptors that may account for discrepancies in patient responses to treatments. The present study commences characterisation of the profiles of human 5-HT₃ receptors containing C and/or E subunits. Recombinant 5-HT₃ receptors were expressed transiently in HEK293T cells and expression was checked via immunocytochemistry staining against each epitope-tagged subunits. Functional characterisation of different combinations of 5-HT₃ receptor complexes was studied via patch clamp whole cell recordings. In this study, increased current was seen in cells containing A and C subunits but only subtle changes were seen in the electrical properties of cells expressing A, AE, or ACE subunits in response to the ligand, 5-HT. Both di- and tri-heteromeric 5-HT₃ receptors were significantly inhibited by the antagonists, ondansetron and palonosetron. Notably, palonosetron exerted stronger and more rapid inhibition on the 5-HT₃ receptor ACE tri-heteromer compared to homomeric and di-heteromeric counterparts. This study demonstrated that the C and E subunits when assembled as simple or complex heteromeric 5-HT₃ receptors may alter efficacies of 5-HT and clinically used antagonists such as ondansetron and palonosetron, and this in turn may have implications for patient responses to therapies.