The influx of neutrophils into the lung is a sentinel event in LPS-induced acute lung inflammation. Previous studies have shown that systemic inhibition of p38 decreases LPS-induced neutrophil influx into the alveolar space but has no effect on pulmonary parenchymal neutrophil accumulation or on microvascular leak, indicating other pathways are important in LPS-induced acute lung inflammation. This study examined the role of c-Jun N-terminal kinase in LPS-induced acute lung inflammation. Systemic inhibition of c-Jun N-terminal kinase, with the specific c-Jun N-terminal kinase inhibitor SP600125, decreased the LPS-induced accumulation of neutrophils into the lung parenchyma and alveolar space. In addition, increases in microvascular leak after LPS exposure were diminished by c-Jun N-terminal kinase inhibition. To determine mechanisms by which systemic c-Jun N-terminal kinase inhibition decreased pulmonary neutrophil influx, LPS and tumor necrosis factor alpha (TNF-alpha-)-induced neutrophil actin assembly and retention were examined. Neutrophil actin assembly was decreased after LPS and TNF-alpha stimulation with SP600125 pretreatment, as well as LPS-induced neutrophil retention. Finally, c-Jun N-terminal kinase inhibition decreased Cdc42 activation after LPS or TNF-alpha stimulation, thereby providing one mechanism by which c-Jun N-terminal kinase inhibition decreased actin assembly, and thereby pulmonary neutrophil accumulation.