To discover selective serotonin 5-HT4 receptor agonists, we designed and synthesized substituted pyrrolizidines 1 using the principle of conformational restriction. Structure-activity relationships were developed, whereby chemical manipulation allows either 5-HT4 agonism or 5-HT3 antagonism to be selectively enhanced. We report herein that compound 1B (SC-53116, 1-S,8-S isomer) surpasses the potency and selectivity of other benzamides (metoclopramide, zacopride, renzapride, cisapride) and the benzimidazolone (BIMU-8) as an agonist at the newly described serotonin 5-HT4 receptor. 1B exhibited potent 5-HT4 agonism (ED50 = 23 nM) in the rat tunica muscularis mucosae preparation, rivaling serotonin, and modest activity at the 5-HT3 receptor (Ki = 152 nM). Unlike metoclopramide and cisapride, 1B showed little affinity (IC50 > 10000 nM) for dopamine D2, 5-HT2, or α1 adrenergic receptors. In conclusion, substituted pyrrolizidines 1 constitute a novel series of potent and selective 5-HT4 agonists, with 1B being the most potent and selective agent reported to date. The in vitro 5-HT4 agonist property of 1B correlates well with potent gastrointestinal prokinetic activity in vivo, and 1B will be a valuable tool for investigating the role of 5-HT4 receptors in various central nervous system and gastrointestinal diseases.