Tumor necrosis factor alpha (TNFalpha), a pro-inflammatory cytokine, enhances the development of pain and hyperalgesia, although the molecular mechanisms are not well understood. This study evaluated the hypothesis that TNFalpha increases the sensitivity of rat trigeminal neurons to capsaicin via two different mechanisms triggered by either brief or sustained exposure to the cytokine. A brief (5 min) application of TNFalpha significantly sensitized capsaicin-evoked accumulation of intracellular calcium ([Ca2+]i) (226.4+/-37.7 nM vs. 167.5+/-31.3 nM) and increased capsaicin-evoked nocifensive behavior (78.3+/-9.7 vs. 30.9+/-3.6 s) as compared with vehicle pretreatment (P<0.01 for both). Sustained (30 min to 4 h) exposure of cultured neurons to TNFalpha evoked a twofold increase in mRNA transcript (P<0.05) and protein levels (P<0.01) of transient potential receptor vanilloid type 1 (TRPV1). This long-term up-regulation of TRPV1 expression by TNFalpha correlated with enhancement in capsaicin-induced calcitonin gene-related peptide release (P<0.05). Demonstration of colocalization of TNFalpha receptor subtypes I and II with TRPV1 in almost all (>90%) TRPV1 expressing neurons provides evidence consistent with a direct interaction on the same subpopulation of sensory neurons. In summary, our data demonstrate that TNFalpha directly enhances the sensitivity of rat trigeminal neurons to capsaicin via both rapid, non-genomic mechanisms as well as sustained genomic regulation in TRPV1 expression. Thus, increased sensitization and up-regulation of TRPV1 constitutes a potential mechanism by which TNFalpha mediates inflammatory hyperalgesia and pain.