In order to establish if the canine 5-hydroxytryptamine type 3A (5-HT3A) receptors share the pharmacological profile with human 5-HT3A receptors, we cloned and performed a molecular pharmacological characterization of the canine 5-HT3A receptor. The 5-HT3A cDNA was cloned from canine brain by polymerase chain reaction amplification. It encodes a 483 amino acid peptide that exhibits from 80% (mice) to 90% (ferrets) identity to other sequenced mammalian 5-HT3A receptors. The receptor agonists 5-hydroxytryptamine (5-HT) and meta-chlorophenylbiguanide (mCPBG) showed little differences between the two species, whereas 2-methyl-5-hydroxytryptamine (2-Me-5-HT) was ten times weaker at canine receptors than at human receptors. The potencies at the canine 5-HT3 receptors were 9.9 μM (5-HT), 79 μM (2-Me-5-HT) and 0.8 μM (mCPBG). The selective, competitive receptor antagonist ondansetron was ten times more potent at human receptors compared to canine receptors (Kb = 0.9 nM), while (+)-tubocurarine was 1000-fold more potent at canine receptors (Kb = 3.0 nM) than at human receptors. Examination of the presumed ligand binding extracellular domain revealed one residue, where the canine receptor differs from all previously characterized 5-HT3A receptors, i.e. other species contain a conserved Trp195, whereas the canine orthologue contains a Leu195. To address the differences in potencies at the human and canine 5-HT3A receptors seen in this study, we introduced a L195W point mutation in the canine orthologue. Data showed that the 195 residue can affect receptor agonist potency and efficacy as well as antagonist potency, but did produce a pharmacological profile identical to the human orthologue. We therefore conclude that position 195 is strongly involved in the receptor–ligand interaction, but additional residues must contribute to the overall pharmacological profile.