Prucalopride is a gastrointestinal prokinetic drug that acts through 5-HT4 receptors, but its potential effects on cardiac atrial function are unknown. We investigated the effects of prucalopride on human right atrium, piglet left atrium, and piglet sinoatrial node. The effects of prucalopride on 5-HT4 receptor splice variants a, b, g and i, known to be expressed in human atrium, were studied for comparison. Prucalopride was an inotropic partial agonist, compared with 5-HT, on paced human atrial trabeculae (-logEC50M=7.4) and porcine left atria (-logEC50M=7.2), with intrinsic activity of 0.77 and 0.63 respectively. Prucalopride (1 microM) surmountably antagonized the positive inotropic effects of 5-HT on human (pK(P)=7.2) and porcine (pK(P)=7.1) atrium. Prucalopride was also a chronotropic partial agonist (-logEC50M=7.4, intrinsic activity=0.72 with respect to 5-HT) on spontaneously beating piglet atria. The cardiostimulant effects of prucalopride were prevented by GR113808 (1 microM), consistent with mediation through 5-HT4 receptors. Prucalopride bound to recombinant 5-HT4(a), 5-HT4(b), 5-HT4(g), and 5-HT4(i) receptors, labeled by [3H]GR113808, with pKi values of 7.6, 7.5, 7.4, and 7.8 respectively. Prucalopride stimulated adenylyl cyclase as a partial agonist on 5-HT4(a), 5-HT4(b), and 5-HT4(i) receptors with intrinsic activities of 0.82, 0.86, and 0.78 and -logEC50 values of 7.2, 7.3, and 7.2 respectively. At the 5-HT4(g) receptor prucalopride acted as a full agonist (-logEC50M=8.0) compared with 5-HT in the cell line tested, which was probably due to high receptor expression levels. We conclude that prucalopride is a cardiostimulatory partial agonist through human and porcine 5-HT4 receptors. Since prucalopride acts similarly through 5-HT4(a), 5-HT4(b), 5-HT4(g), and 5-HT4(i) receptors, any of these variants could be involved in the mediation of cardiostimulation.