Activator protein 1 (AP-1) binds to the promoters of many genes involved in immune and inflammatory responses. We have previously shown that the p38 mitogen-activated protein (MAP) kinase regulates NF-κB-dependent gene expression by modulating the phosphorylation and subsequent activation of TATA-binding protein (TBP). In this study, we asked whether the p38 MAP kinase regulated the transcriptional activity of AP-1. We found that phorbol 12-myristate 13-acetate (PMA) was unable to drive the AP-1-dependent reporter gene in THP-1 cells. PMA activated both the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase MAP kinases, but it did not activate the p38 MAP kinase. We found that cells expressing MAP kinase kinase 6(Glu), which is the upstream kinase that activates the p38 MAP kinase, had significantly increased AP-1-dependent gene expression alone and when stimulated with PMA. These cells also had increased phosphorylation of native c-Jun, suggesting that both c-Jun NH2-terminal kinase and p38 MAP kinases phosphorylate c-Jun. More importantly, expression of a constitutive active MAP kinase kinase 6(Glu) resulted in the phosphorylation of a His-TBP fusion protein and increased direct interaction of TBP with c-Jun. These findings suggest that in macrophages, the p38 MAP kinase regulates AP-1-driven transcription by modulating the activation of TBP.