The present study was designed to investigate which subtypes of spinal 5-HT receptors are involved in 5-HT-induced antinociception using the mechanical pain test. Serotonin and various selective antagonists or agonists for 5-HT receptor subtypes (5-HT_1A, 5-HT_1B, 5-HT_2A, 5-HT_2C, 5-HT₃ and 5-HT₄) were administered intrathecally (i.t.) in rats. The i.t. injection of 5-HT (1 μg) produced significant antinociceptive effects using the paw pressure test. Pretreatment with the 5-HT_2C receptor antagonist mesulergine (1 and 10 μg) and the 5-HT₃ receptor antagonist tropisetron (1 and 10 μg) reversed totally the antinociception induced by 5-HT. Furthermore, at a dose of 10 μg, both the 5-HT_2A receptor antagonist ketanserin and the 5-HT_1B receptor antagonist penbutolol, but neither the 5-HT_1A receptor antagonist WAY 100635 nor the 5-HT₄ receptor antagonist GR113808, attenuated the antinociceptive effect induced by 5-HT. In addition, an i.t. injection of the 5-HT₃ agonist mCPBG induced significant antinociceptive effects whereas the 5-HT₂ agonist DOI did not produce analgesia. These results suggest that although the precise degree of the involvement of spinal serotonergic 5-HT₃ receptors remains to be elucidated due to some differences in the effect of agonists or antagonists, these receptors seem to play a role in the antinociceptive effect of 5-HT against a mechanical acute noxious stimulus. The involvement of 5-HT_2C is more questionable due to the observed discrepancies between the effects of the used agonist and antagonist. 5-HT_1A and 5-HT₄ receptors do not seem to be involved. In addition, a possible functional interaction between spinal serotonergic receptors may exist.