The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-alpha synthesis is still a matter of controversy. Therefore, we focused our study on TNF-alpha protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-alpha might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation.