In this study, we have investigated serotonin hyperalgesia employing the mechanical paw withdrawal nociceptive threshold test in the rat. Intradermally injected serotonin was found to produce a dose-dependent hyperalgesia that was not attenuated by procedures which eliminate the known indirect mechanisms of hyperalgesia such as sympathectomy, polymorphonuclear leukocyte depletion or cyclooxygenase inhibition. In addition, the latency to onset of serotonin hyperalgesia is extremely short, with maximal hyperalgesia observed in less than 1 min, a similar temporal onset to direct-acting hyperalgesic agents such as prostaglandin E2. The results suggest, therefore, that the hyperalgesic effects of serotonin in our animal model are exerted by direct action on primary afferent neurons. Only the intradermal injection of selective serotonin (5-hydroxytryptamine; 5-HT) agonists for the 1A receptor subset (5-HT1A), (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide and N,N-dipropyl-5-carboxamido-tryptamine maleate, produced dose-dependent hyperalgesia. No hyperalgesia was seen after 5-HT1B, CGS-12066B maleate and m-trifluoromethylphenyl-piperazine hydrochloride; 5-HT2+IC, alpha methyl 5HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl; or 5-HT3, 2-methyl-5-hydroxytryptamine maleate and phenylbiguanide, agonists. Similarly, only the 5-HT1A antagonists, spiroxatrine and spiperone, attenuated the hyperalgesia induced by intradermally injected serotonin. 5-HT2+IC antagonists, mesulergine and ketanserin, and 5-HT3 antagonists, quipazine and 3-tropanyl-indole-3-carboxylate, did not significantly attenuate 5-HT hyperalgesia. We conclude that serotonin produces hyperalgesia by a direct action on the primary afferent neuron via the 5-HT1A subset of serotonin receptors.