The physiological effect of serotonin (5-HT) is mediated in the central and periphery systems by seven subtypes of receptors. All but one are members of the G protein coupled receptor (GPCRs) family. Thirteen genes coding for the 5-HT GPCRs have been characterized, and only three 5-HT receptors (5-HT4R, 5-HT6R, and 5-HT7R) are coupled to Gs proteins and stimulate adenylyl cyclase activity, giving rise to an increase of the intracellular cAMP. Considerable interest has been devoted to the characterization of 5-HT4 receptors since their discovery in mouse colliculi neuronal cells and in the guinea pig ileum. Their presence in the gut allowed the proposal of a mechanism responsible for the pharmacological activity of several gastrokinetic benzamide drugs, such as metoclopramide, zacopride, and renzapride. Several splice variants of the 5-HT4 receptors have been cloned, and they differ in the length of their C-terminal ends. When studied in heterologous expression systems, these receptors present some functional and pharmacological differences, with the length of the C-terminal sequence implicated in constitutive activity. 5-HT4 receptors are localized in the central nervous system, heart, intestine, adrenal cortex, and bladder, mediating important physiological processes including acetylcholine release in the hippocampus, increased Ca2+ and pacemaker currents in the atrium, initiation of intestinal peristaltic reflex, and enhanced corticosterol release in the adrenal gland. Consequently, 5-HT4 receptors are implicated in various pathological disorders and constitute a valuable target for new drug design. Advances have been made in developing molecules for irritable bowel syndrome (IBS), characterized by altered bowel function due to intestinal serotoninergic dysfunction, and other promising avenues include preventing atrial fibrillation with 5-HT4 antagonists, improving cognitive functions via 5-HT4 agonists enhancing hippocampal cholinergic transmission, and treating voiding disorders associated with detrusor hypocontractility. The first generation of 5-HT3 receptor antagonist benzamides, which act as ligands for both 5-HT3 and 5-HT4 receptors, played a major role in discovering potent and selective 5-HT4 ligands. Previous reviews have covered structure-activity relationships (SARs) in this field. The present paper describes recent advances in medicinal chemistry, mechanisms explaining the physiological role of 5-HT4 receptors in different tissues, and putative clinical applications of 5-HT4 agonists and antagonists.