Prucalopride is a novel enterokinetic compound and is the first representative of the benzofuran class. We set out to establish its pharmacological profile in various receptor binding and organ bath experiments. Receptor binding data have demonstrated prucalopride's high affinity to both investigated 5-HT₄ receptor isoforms, with mean pK_i estimates of 8.60 and 8.10 for the human 5-HT_4a and 5-HT_4b receptor, respectively. From the 50 other binding assays investigated in this study only the human D₄ receptor (pK_i 5.63), the mouse 5-HT₃ receptor (pK_i 5.41) and the human σ₁ (pK_i 5.43) have shown measurable affinity, resulting in at least 290-fold selectivity for the 5-HT₄ receptor. Classical organ bath experiments were done using isolated tissues from the rat, guinea-pig and dog gastrointestinal tract, using various protocols. Prucalopride was a 5-HT₄ receptor agonist in the guinea-pig colon, as it induced contractions (pEC₅₀=7.48±0.06; insensitive to a 5-HT_2A or 5-HT₃ receptor antagonist, but inhibited by a 5-HT₄ receptor antagonist) as well as the facilitation of electrical stimulation-induced noncholinergic contractions (blocked by a 5-HT₄ receptor antagonist). Furthermore, it caused relaxation of a rat oesophagus preparation (pEC₅₀=7.81±0.17), in a 5-HT₄ receptor antagonist sensitive manner. Prucalopride did not cause relevant inhibition of 5-HT_2A, 5-HT_2B, or 5-HT₃, motilin or cholecystokinin (CCK₁) receptor-mediated contractions, nor nicotinic or muscarinic acetylcholine receptor-mediated contractions, up to 10 μM. It is concluded that prucalopride is a potent, selective and specific 5-HT₄ receptor agonist. As it is intended for treatment of intestinal motility disorders, it is important to note that prucalopride is devoid of anti-cholinergic, anticholinesterase or nonspecific inhibitory activity and does not antagonise 5-HT_2A, 5-HT_2B and 5-HT₃ receptors or motilin or CCK₁ receptors.