This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT₄ receptor splice variants [h5-HT_4(a), h5-HT_4(b), h5-HT_4(c), h5-HT_4(d) and h5-HT_4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT₄ benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pK_i: 7.38–7.91) ≧ Y-36912 (pK_i: 7.03–7.85) = BIMU 1 (pK_i: 6.92–7.78) ≧ DAU 6236 (pK_i: 6.79–7.99) ≧ 5-HT (pK_i: 5.82–7.29) ≧ 5-MeOT (pK_i: 5.64–6.83) ≧ renzapride (pK_i: 4.85–5.56). We obtained affinity values for the 5-HT_4(b), (d) and (g) variants for RS67333 (pK_i: 7:48–8.29), prucalopride (pK_i: 6.86–7.37) and zacopride (pK_i: 5.88–7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically.