A critical feature of sepsis-induced acute lung injury is the release of cytokines from endotoxin (LPS)-stimulated alveolar macrophages (AM). LPS is also known to activate various members of the mitogen-activated protein kinase (MAPK) family in other types of cells. In this study, we evaluated whether multiple members of the MAPK family regulate cytokine gene expression in LPS-stimulated AM. We found that LPS activates both the extracellular signal-regulated kinase (Erk) and p38 kinases, and that this activation is augmented when the cells are cultured in serum. Inhibition of either the Erk (with PD98059) or p38 (with SB203580) kinase pathway resulted in only a partial reduction in cytokine (interleukin-6 and tumor necrosis factor) messenger RNA accumulation and cytokine release, whereas inhibition of both pathways simultaneously resulted in a decrease in cytokine gene expression to near-control levels. Nuclear run-on assays showed that the effect of these MAPK pathways on LPS-induced expression of the cytokine genes was attributable, at least in part, to regulation of gene transcription. These findings suggest that activation of both the Erk and p38 kinase pathways is necessary for optimal cytokine gene expression in LPS-stimulated human AM, and that the MAPK pathways play a critical role in the inflammatory response that occurs in sepsis-induced acute lung injury.