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The present study was designed to investigate which subtypes of 5-HT receptors are involved in 5-HT-induced hyperalgesia using behavioral assessment of hyperalgesia. 5-HT and various putative agonists for 5-HT receptor subtypes (5-HT1A, 2, 3) were intradermally injected into the rat ipsilateral hindpaw. Paw-withdrawal latency to radiant heat stimulation was examined every 15 min for 2 h. Injection of 5-HT (30 μg) and 5-HT2A receptor agonist (α-methyl 5-HT; 0.86 mg/kg) significantly reduced the paw-withdrawal latency. On the other hand, injection of 5-HT3 receptor agonists (2-methyl 5-HT; 0.86 mg/kg, m-CPG; 8 mg/kg) did not produce hyperalgesia. Furthermore, pre-treatment with 5-HT2A receptor antagonist (ketanserin), but not with 5-HT3 receptor antagonist (tropisetron), attenuated the behavioral response after the injection of 5-HT. These findings strongly suggest that the 5-HT2A receptor subtype, but not the 5-HT3 subtype, is involved in 5-HT-induced hyperalgesia in acute injury and inflammation in the rat. In situ hybridization histochemistry revealed the presence of 5-HT2 receptor mRNA in a subpopulation of both large and small neurons in the rat dorsal root ganglia.