In the present study, binding affinities of 5-hydroxytryptamine-4 (5-HT₄) ligands for the human 5-HT_4d receptor were determined using the agonist [³H]5-HT and the selective 5-HT₄ antagonist [³H]GR113,808. We also compared the affinity differences between [³H]5-HT binding (K_H) and [³H]GR113,808 binding (K_L) with their activities as 5-HT₄ ligands. Binding studies using [³H]5-HT revealed that the human 5-HT_4d receptor has two binding sites, whereas [³H]GR113,808 yielded a single binding site. Additionally, the number of [³H]5-HT binding sites decreased in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPγS), but the number of [³H]GR113,808 sites did not change. In competitive binding assays, full agonists such as 5-methoxytryptamine and tegaserod showed 2- to 8-fold higher affinities for [³H]5-HT binding (K_H) than for [³H]GR113,808 binding (K_L) (K_H<K_L). Conversely, antagonists showed lower affinities for [³H]5-HT binding than for [³H]GR113,808 binding (K_H>K_L). Finally, partial agonists displayed similar binding affinities for both radioligands (K_H = K_L). These findings suggest that the equilibrium between active and inactive states of the human 5-HT_4d receptor relies on the functional activities of 5-HT₄ ligands, and these states affect the affinities of 5-HT₄ ligands in the competitive binding assay.