The aim of this study was to demonstrate the presence of 5-HT3 receptors in the mouse bladder and to determine their location. Bladder strips from female mice were set up in gassed Krebs-Henseleit solution at 37°C and contractions recorded in response to electrical field stimulation (8 Hz, 60 V, 0.5-ms pulse duration) applied for 2 s every 50 s. The potentiating effects of 5-hydroxytryptamine (5-HT) were recorded (in the presence of 1-μM methysergide and 1-μM GR125487 to isolate the 5-HT3 receptor response), and contractions were expressed as a percentage of the response to 0.1-M KCl. Responses to (5-HT) were also obtained in the presence of the 5-HT3 receptor antagonist, ondansetron. RT-PCR was used to detect the expression of the 5-HT3A and 5-HT3B subunit transcripts of the mouse 5-HT3 receptor. 5-HT and 5-HT3 receptor agonists caused concentration-dependent increases in the force of neurogenic contractions without affecting the baseline tone. The rank order of potency was: meta-chloro-phenylbiguanide (m-CPB) = 5-HT > 2-methyl-5-HT (2m5-HT) = 1-phenylbiguanide (1-PBG). The respective pEC50 values were: 6.42 ± 0.2 = 5.95 ± 0.19 > 5.35 ± 0.12 = 5.14 ± 0.13. m-CPB acted as a full agonist (E max = 40.65 ± 3.81% KCl), but both 2m5-HT and 1-PBG acted as lower potency partial agonists. Ondansetron (30, 100, 300 nM) caused concentration-related rightward displacements to the concentration-effect curve to 5-HT. Nonlinear regression analysis of the effect of the ondansetron concentrations on the pEC50 values produced a pKB value of 8.29 ± 0.22. Desensitization of sensory nerves to the contractile effect of capsaicin (10 μM for 60 min) did not alter the ability of 5-HT to potentiate neurogenic contractions. 5-HT (3 μM) inhibited contractions induced by direct muscle stimulation (lignocaine, 300 μM and 10-ms pulse width). m-CPB also caused the same effect with a pIC50 of 6.62 ± 0.10 and an E max of 48.03 ± 2.25%. The concentration-response curve to m-CPB was shifted rightwards by ondansetron (1 μM) giving an apparent pKB value of 8.15 ± 0.33. mRNA for both the 5-HT3A and 5-HT3B receptor subunits was detected in the detrusor as well as the mucosa with a greater relative expression of the 5-HT3A subunit in both layers. This study demonstrates that 5-HT mediates enhanced neurogenic contractions of the mouse bladder muscle by an action at 5-HT3 receptors located prejunctionally on nonsensory nerve elements. Additionally, an inhibitory postjunctional population of the 5-HT3 receptor was identified. The presence of the 5-HT3 receptor was confirmed by the expression of both 5-HT3A and 5-HT 3B receptor subunits of the 5-HT3 receptor.