The pharmacological properties of the (R) and (S) enantiomers of RS 56532 have been studied in vitro and in vivo. In radioligand binding studies at 5-HT 4 receptors in guinea-pig striatum, (S) RS 56532 exhibited a higher affinity than (R) RS 56532 (− log i = 7.6 and 6.5, respectively). (S) RS 56532 acted as a potent agonist at 5-HT 4 receptors mediating relaxation of rat oesophageal muscularis mucosae (−log EC 50 = 7.9) while (R) RS 56532 acted as a weaker agonist at this receptor (− log EC 50< 6.0). These data suggest that at 5-HT 4 receptors, the enantiomeric selectivity of RS 56532 was (S) > (R). In binding studies at 5-HT 3 receptors in rat cortex, (R) RS 56532, conversely, exhibited a higher affinity than (R) RS 56532 (− log K i = 9.1 and 8.0, respectively). At 5-HT 3 receptors in guinea-pig isolated ileum, (R) RS 56532 exhibited an affinity (− log K B) of 7.9, whereas (S) RS 56532 (1 nM–1 μM) was inactive. No agonism was observed at ileal 5-HT 3 receptors with either enantiomers. These data suggest that at 5-HT 3 receptors in rat and guinea-pig, both enantiomers acted as antagonists, with (R) > (S) RS 56532. At the non-5-HT 3, high affinity ‘(R) zacopride’ site, (R) RS 56532 exhibited a higher affinity than (S) RS 56532 (− log K i = 6.1 and 4.9). This site was insensitive to potent 5-HT 3 antagonists such as (R) YM 060 or ondansetron. However, it was recognized with relatively high affinity (− log K i = 7.5) by the (R), but not (S)d enantiomer, of RS 42358 (− log K i = 4.7). Since (S) RS 42358 is a high affinity 5-HT 3 receptor antagonist, these data further highlight the dissimilarity between the 5-HT 3 receptor and the ‘(R) zacopride’ site. The ‘(R) zacopride’ site also appeared to be pharmacologically distinct from the 5-HT 4 receptor, since 5-HT 4 ligands such as renzapride, SDZ 205,557 or RS 23597-190 exhibited low affinities. The enantiomeric selectivity of (R) and (S) RS 56532 in vivo was consistent with findings in vitro. At 5-HT 4 receptors mediating tachycardia in the pig, 5-HT induced a dose-dependent tachycardia (ED 50 = 3μg kg −1, i.v.; maximum response = 90–100 beats min −1). (S) RS 56532 increased heart rate by 88 min −1 with a potency of (ED 50) of 3 μg kg −1, i.v. In contrast, a tachycardia effect (23 beats min −1) of (R) RS 56532 was seen only at 1 mg kg −1, i.v. (R) RS 56532 was more potent than (S) RS 56532 (ID 50 = 3 and 78 μg kg −1, i.v. respectively) at inhibiting the von Bezold Jarisch reflex, a response mediated by 5-HT 3 receptor activation. Similarly, (R) RS 56532, at 0.1 mg kg −1 p.o., inhibited cisplatin induced emesis in the ferret, from 19.8 to 5.8 emetic episodes. In contrast, (S) RS 56532 was inactive at this oral dose. The emetic response to neoplastic agents such as cisplatin is also mediated by 5-HT 3 activation. In summary, RS 56532 in vitro and in vivo, exhibits opposing enantiomeric selectivity at 5-HT 3 and 5-HT 4 receptors, i.e. 5-HT 3—(R) > (S); 5-HT 4—(S) > (R). The affinity of the (R) enantiomer at 5-HT 3 receptors and the potency of the (S) enantiomer at 5-HT 4 receptors render them useful pharmacological tools to further define the binding domains of these two 5-HT receptor subtypes. Furthermore, these data show that 1,8-naphthalimides, such as (S) RS 56532, represent a novel class of potent 5-HT 4 receptor agonists.