Repeated inflammation in the periphery is a major cause of chronic inflammatory pain. We have showed that blockade of 5-HT2A receptors in the periphery inhibits repeated inflammation-induced pain hypersensitivity. The present study investigated whether inhibition of 5-HT2A receptor signaling at the site of inflammation could inhibit repeated inflammation-induced neurochemical changes in spinal dorsal horn neurons. Treatment with the 5-HT2A receptor antagonist ketanserin (20mug) in the hindpaw following intraplantar injection of carrageenan inhibited the increase in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity, a marker of nitric oxide synthase (NOS), in the spinal dorsal horn. Administration of formalin (1%) in the hindpaw following the carrageenan injection evoked a great increase in NADPH-d reactivity in spinal dorsal horn neurons on the ipsilateral and contralateral sides. These changes were abolished by the pretreatment of ketanserin (20mug). The ketanserin treatment also reduced the repeated inflammation-induced expression of protein kinase gamma (PKCgamma) in the membrane of spinal dorsal horn neurons and increased PKCgamma protein level in the cytosol. Following the treatment with the opioid receptor antagonist naloxone methiodide (5mg/kg, s.c.), the administration of ketanserin failed to inhibit the repeated inflammation-induced increase in NADPH-d reactivity and c-Fos expression in the spinal dorsal horn. These results suggest that 5-HT2A receptor bioactivity in the inflammatory site plays an important role in repeated inflammation-induced central sensitization. The present study supports the notion that targeting 5-HT2A receptors in the periphery can be a promising therapy for relieving chronic inflammatory pain.