Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT₃) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT₃ receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT₃ receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT₃ receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT₃ receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.