Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1β cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1β protein and steady state message in a dose-dependent manner in both RAW264. 7 and J774 cell lines. The loss of IL-1β message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an∼ 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-α message. LPS-stimulated p38 MAPK activity in the RAW264. 7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor (s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-κB elements were only partially inhibited (∼ 35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-κB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1β transcription by acting through C/EBP/NFIL-6 transcription factors.