Melatonin has been shown to be produced by nonpineal cells and possess anti-inflammatory actions in animal models. In the present study, we tested the hypothesis that melatonin suppresses the expression of proinflammatory genes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (INOS) by a common transcriptional mechanism. Melatonin but not tryptophan or serotonin inhibited lipopolysaccharide (LPS)-induced COX-2 and iNOS protein levels and promoter activities in RAW 264.7 cells in a time- and concentration-dependent manner. LPS or LPS plus interferon-gamma (IFNgamma) increased binding of all 5 isoforms of NF-kappaB to COX-2 and iNOS promoters. Melatonin selectively inhibited p52 binding without affecting p100 expression, p52 generation from p100, or p52 nuclear translocation. p52 acetylation was enhanced by LPS, which was abrogated by melatonin. Melatonin inhibited p300 histone acetyltransferase (HAT) activity and abrogated p300-augmented COX-2 and iNOS expression. HAT inhibitors suppressed LPS-induced p52 binding and acetylation to an extent similar to melatonin, and melatonin did not potentiate the effect of HAT inhibitors. These results suggest that melatonin inhibits COX-2 and iNOS transcriptional activation by inhibiting p300 HAT activity, thereby suppressing p52 acetylation, binding, and transactivation.