Prostaglandin J₂ (PGJ₂) and its metabolites Δ¹²-PGJ₂ and 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) are naturally occurring derivatives of prostaglandin D₂ that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ₂ is a high-affinity ligand for the peroxisome proliferator-activated receptor γ (PPARγ) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor α, in a PPARγ-dependent manner. We report here that 15d-PGJ₂ potently inhibits NF-κB-dependent transcription by two additional PPARγ-independent mechanisms. Several lines of evidence suggest that 15d-PGJ₂ directly inhibits NF-κB-dependent gene expression through covalent modifications of critical cysteine residues in IκB kinase and the DNA-binding domains of NF-κB subunits. These mechanisms act in combination to inhibit transactivation of the NF-κB target gene cyclooxygenase 2. Direct inhibition of NF-κB signaling by 15d-PGJ₂ may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.