Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. We have previously reported that Cdk5 participates in the regulation of nociceptive signaling, and the expression of Cdk5 and its activator, p35, are up-regulated in nociceptive neurons during peripheral inflammation. The aim of our current study was to identify the proinflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that tumor necrosis factor-alpha (TNF-alpha) activates p35 promoter activity in a dose- and time-dependent manner and concomitantly up-regulates Cdk5 activity. Because TNF-alpha is known to activate ERK1/2, p38 MAPK, JNK, and NF-kappaB signaling pathways, we examined their involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK, and NF-kappaB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of Egr-1, a transcription factor, were increased by TNF-alpha treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which carrageenan injection into the hind paw causes hypersensitivity to heat stimuli, TNF-alpha mRNA was increased at the site of injection. These findings suggest that TNF-alpha-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling.