5-Hydroxytryptamine (5-HT)(3) receptors have been proposed to modulate nociception and pain responses at the spinal level. To gain insight into the cellular mechanism of 5-HT(3) receptors, we examined their expression in GABAergic and enkephalinergic (ENKergic) neurons in the spinal dorsal horn (SDH) using single-cell reverse transcription-polymerase chain reaction (RT-PCR). The glutamic acid decarboxylase (GAD)(67)-green fluorescent protein (GFP) knock-in mouse was used in which all GABAergic neurons were fluorescent. The general tissue RT-PCR results showed that 5-HT(3A) receptor subunit mRNA was present in the mouse SDH, while 5-HT(3B) receptor subunit was absent. Single-cell RT-PCR results showed that 76.2% (16/21) and 33.3% (7/21) of the total 5-HT(3A)-expressing neurons were positive for GAD(67) and preproenkephalin (PPE, a precursor of ENK), respectively. 5-HT(3A) receptor subunit was detected in 28.1% (16/57) of GABAergic neurons and 22.6% (7/31) of ENKergic neurons. About 40.4% (23/57) of GABAergic neurons expressed PPE mRNA. Of the neurons that co-express GAD(67) mRNA and PPE mRNA, about 22% expressed 5-HT(3A) mRNA. These observations indicate that 5-HT(3A) receptor co-localizes with GABA and ENK in the SDH, suggesting that serotonin may activate GABAergic and ENKergic neurons via 5-HT(3A) receptor subunit and therefore affect the release of GABA and ENK. The different cellular localization of 5-HT(3A) receptor subunit suggest the complex participation of this receptor in the inhibitory neuronal circuits of the SDH neurons.