Some transcription factors involved in the regulation of cyclooxygenase 2 (COX-2) expression in macrophage, including NF-kappaB, interact with p300, which contains histone acetyltransferase (HAT) enzyme complex. Chromatin structure is regulated by modifying enzymes, including HAT, and plays an important role in eukaryotic gene regulation through histone modification. We hypothesized that changes in chromatin structure related to phosphorylation and acetylation of histone H3 adjacent to key DNA binding sequence motif in the COX-2 promoter contribute to COX-2 gene activation in macrophages. Sodium butyrate (NaBT) is a short-chain fatty acid that possesses histone deacetyltransferase-inhibiting activity. Our data show that NaBT accentuates LPS-induced COX-2 gene expression at a transcriptional level, even though NaBT alone does not induce the COX-2 gene expression. Using a chromatin immunoprecipitation assay, we showed that costimulation of RAW 264.7 cells with NaBT and LPS synergistically increases COX-2 gene expression through both acetylation and phosphorylation of histone H3 at the promoter site. Our data show that NaBT accentuates LPS-induced COX-2 gene expression through MAP kinase-dependent increase of phosphorylation and acetylation of histone H3 at the COX-2 promoter site. These data indicate that posttranslational modification of histone H3 has a major effect on COX-2 gene expression by macrophages.