With the central effects of serotonin (5-HT) on ejaculation having been relatively established, we investigated the peripheral effects of serotonin on the contractile responses of rat seminal vesicles and vasa deferentia. Male Sprague-Dawley rats were grouped on the basis of the agents administered: serotonin, clomipramine, or fluoxetine. The intraluminal pressures of the seminal vesicles and of the vasa deferentia were measured simultaneously. Control responses to hypogastric nerve stimulation (HNS) were recorded in each animal, and HNS was repeated after drug administration. Expression of the mRNAs of the 5-hydroxytryptamine (5-HT) receptors (5-HT1A, 5-HT1B, and 5-HT2C), which have been suggested to be involved in the ejaculation process, were examined by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Serotonergic agents resulted in the concentration-dependent inhibition of HNS-induced seminal vesicle pressure increases (clomipramine > serotonin > fluoxetine). Vasal pressure responses were effectively inhibited by clomipramine and serotonin, but fluoxetine had no effect. No significant difference was observed in the relative expression levels of 5-HT1A receptor mRNA in seminal vesicles and in the vasa deferentia. However, the expression levels of 5-HT1B and 5-HT2C receptor mRNAs were lower in the vasa deferentia than in the seminal vesicles. These in vivo and in vitro experimental results provide evidence for the peripheral role of 5-HT in the regulation of contractile responses of the seminal tract. Regional differences in the distribution of the 5-HT receptor subtypes of the seminal vesicles and the vasa deferentia might contribute to the different responses to serotonergic agents shown by these organs.