Insight into the mechanism(s) by which ambient air particulate matter (PM) mediates adverse health effects is needed to provide biological plausibility to epidemiological studies demonstrating associations between PM exposure and increased morbidity and mortality. Although in vitro PM studies provide an understanding of mechanisms by which PM affects pulmonary cells, it is difficult to extrapolate from in vitro to in vivo mechanisms of PM-induced lung injury. We examined in vivo mechanisms of lung injury generated by oil combustion particles. Rats were pretreated with dimethylthiourea (DMTU) before intratracheal instillation of residual oil fly ash (ROFA). Animals were examined by bronchoalveolar lavage for biomarkers of lung injury, and lung tissues were examined by immunohistochemical, biochemical, and molecular approaches to identify ROFA-induced alterations in intracellular signaling pathways and proinflammatory gene expression. Significant increases in pulmonary inflammation, cytotoxicity, activation of ERK mitogen-activated protein kinase (MAPK), and increases in mRNA levels encoding macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, MCP-1 and matrilysin were observed. DMTU pretreatment inhibited ROFA-induced pulmonary inflammation, cytotoxicity, ERK MAPK activation, and cytokine gene expression. Our findings provide coherence with in vitro PM mechanistic information, allow direct in vitro to in vivo extrapolation, and demonstrate a critical role for oxidative stress in ROFA-induced lung injury and associated molecular pathology.