The present study examined the contribution of 5-hydroxytryptamine (5-HT) to acute peripheral inflammatory pain in rats. We used formalin test in this study. After formalin injection into the rat hind paw, biphasic pain-related behavior (phases 1 and 2) was observed. A microdialysis study revealed that 5-HT was released into the formalin injection site in a formalin concentration-dependent manner (1.25-5%), and its peak time was 18min after the injection. Previous studies suggest that peripheral 5-HT2 receptors are involved in inflammatory pain. Therefore, we next examined whether 5-HT2A and 5-HT2C receptors are involved, and from where 5-HT is released in the formalin test. Local pretreatment with a selective 5-HT2A receptor antagonist, ketanserin, and selective 5-HT2C receptor antagonists, RS102221 and SB242084, inhibited the number of flinches in early part of phase 2 (phase 2A) of the formalin test in a dose-dependent manner. Peripheral pretreatment with sodium cromoglycate (cromolyn), a mast cell membrane stabilizer, completely suppressed 5-HT release and inhibited phase 2 responses of the formalin test. These drugs inhibited c-fos expression in the superficial layer of the spinal dorsal horn of segments L4-5 at 2h after formalin injection. These results indicate that 5-HT released into peripheral tissue and its receptors, 5-HT2A as well as 5-HT2C, at the periphery have an important role in pain-related behaviors during acute peripheral inflammation.