The transcriptional factor p53 has primarily been characterized for its central role in the regulation of oncogenesis. A reciprocal relationship between the activities of p53 and NF-κB has been demonstrated in cancer cells, but there is little information concerning interactions between p53 and NF-κB in inflammatory processes. In this study, we found that neutrophils and macrophages lacking p53, i.e., p53-/-, have elevated responses to LPS stimulation compared with p53+/+ cells, producing greater amounts of proinflammatory cytokines, including TNF-α, IL-6, and MIP-2, and demonstrating enhanced NF-κB DNA-binding activity. p53-/- mice are more susceptible than are p53+/+ mice to LPS-induced acute lung injury (ALI). The enhanced response of p53-/- cells to LPS does not involve alterations in intracellular signaling events associated with TLR4 engagement, such as activation of MAPKs, phosphorylation of IκB-α or the p65 subunit of NF-κB, or IκB-α degradation. Culture of LPS-stimulated neutrophils and macrophages with nutlin-3a, a specific inducer of p53 stabilization, attenuated NF-κB DNA-binding activity and production of proinflammatory cytokines. Treatment of mice with nutlin-3a reduced the severity of LPS-induced ALI. These data demonstrate that p53 regulates NF-κB activity in inflammatory cells and suggest that modulation of p53 may have potential therapeutic benefits in acute inflammatory conditions, such as ALI. Copyright © 2009 by The American Association of Immunologists, Inc.