The role of the 5-HT₃ receptors in pudendal neuromodulation of bladder activity and its interaction with opioid receptors were investigated in anesthetized cats. The bladder was distended with either saline to induce normal bladder activity or with 0.25% acetic acid (AA) to induce bladder overactivity. Pudendal afferent nerves were activated by 5 Hz stimulation at multiples of the threshold (T) intensity for inducing anal twitching. AA irritation significantly reduced bladder capacity to 16.5±3.3% of saline control capacity, while pudendal nerve stimulation (PNS) at 1.5-2T and 3-4T restored the capacity to 82.0±12% (P=0.0001) and 98.6±15% (P<0.0001), respectively. Cumulative doses (1-3mg/kg, i.v.) of ondansetron, a 5-HT₃ receptor antagonist, eliminated low intensity (1.5-2T) PNS inhibition and reduced high intensity (3-4T) PNS inhibition of bladder overactivity. During saline distention, PNS at 1.5-2T and 3-4T significantly increased bladder capacity to 173.2±26.4% (P=0.036) and 193.2±22.5% (P=0.008), respectively, of saline control capacity, but ondansetron (0.003-3mg/kg, i.v.) did not alter PNS inhibition. Ondansetron (0.1-3 mg/kg) also significantly (P<0.05) increased control bladder capacity (50-200%) during either AA irritation or saline distention. In both conditions the effect of low and high intensity PNS were not significantly different. After ondansetron (3 mg/kg) treatment, naloxone (1 mg/kg, i.v.) significantly (P<0.05) decreased control bladder capacity (40-70%) during either AA irritation or saline distention, but failed to affect PNS inhibition. This study revealed that activation of 5-HT₃ receptors has a role in PNS inhibition of bladder overactivity. It also indicated that 5-HT₃ receptor antagonists might be useful for treating overactive bladder symptoms.