Endotoxin-induced cytokine gene expression is regulated, in part, by NF-κB. We have shown Chat both the ERK and p38 mitogen-activated protein (MAP) kinases are necessary for cytokine gene transcription and that the p38 MAP kinase is required for NF-κB-driven transcription, so we hypothesized that the MEK → ERK pathway regulated NF-κB-driven transcription as well. We found that a constitutive active MEK → ERK pathway inhibited NF-κB-driven transcription. In addition, both PD 98059 and a dominant negative ERK2 augmented NF-κB-driven transcription; however, neither PD 98059 nor MEK1 altered NF-κB activation at any level. The constitutive active MEK → ERK pathway inhibited the phosphorylation of TBP, which is necessary for both interaction with ReLA and binding to the TATA box. Due to the fact that we have shown that the p38 MAP kinase modulates TBP activation, we evaluated the effect of the constitutive active MEK → ERK pathway on p38 MAP kinase activity. We found that the MEK → ERK pathway negatively regulates NF-κB-driven transcription, in part, by inhibiting p38 MAP kinase activity. Thus, the ERK and p38 MAP kinases have differential effects on NF-κB-driven transcription.