Serotonin (5-hydroxytryptamine, 5-HT) increases contractile force and elicits arrhythmias through 5-HT₄ receptors in porcine and human atrium, but its ventricular effects are unknown. We now report functional 5-HT₄ receptors in porcine and human ventricle. 5-HT₄ mRNA levels were determined in porcine and human ventricles and contractility studied in ventricular trabeculae. Cyclic AMP-dependent protein kinase (PKA) activity was measured in porcine ventricle. Porcine and human ventricles expressed 5-HT₄ receptor mRNA. Ventricular 5-HT_4(b) mRNA was increased by four times in 20 failing human hearts compared with five donor hearts. 5-HT increased contractile force maximally by 16% (EC₅₀=890 nM) and PKA activity by 20% of the effects of (−)-isoproterenol (200 μM) in ventricular trabeculae from new-born piglets in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine. In ventricular trabeculae from adult pigs (3-isobutyl-1-methylxanthine present) 5-HT increased force by 32% (EC₅₀=60 nM) and PKA activity by 39% of (−)-isoproterenol. In right and left ventricular trabeculae from failing hearts, exposed to modified Krebs solution, 5-HT produced variable increases in contractile force in right ventricular trabeculae from 4 out of 6 hearts and in left ventricular trabeculae from 3 out of 3 hearts— range 1–39% of (−)-isoproterenol, average 8%. In 11 left ventricular trabeculae from the failing hearts of four β-blocker-treated patients, pre-exposed to a relaxant solution with 0.5 mM Ca²⁺ and 1.2 mM Mg²⁺ followed by a switch to 2.5 mM Ca²⁺ and 1 mM Mg²⁺, 5-HT (1–100 μM, 3-isobutyl-1-methylxanthine present) consistently increased contractile force and hastened relaxation by 46% and 25% of (−)-isoproterenol respectively. 5-HT caused arrhythmias in three trabeculae from 3 out of 11 patients. In the absence of phosphodiesterase inhibitor, 5-HT increased force in two trabeculae, but not in another six trabeculae from 4 patients. All 5-HT responses were blocked by 5-HT₄ receptor antagonists. We conclude that phosphodiesterase inhibition uncovers functional ventricular 5-HT₄ receptors, coupled to a PKA pathway, through which 5-HT enhances contractility, hastens relaxation and can potentially cause arrhythmias.