It is well established that p300 plays an important role in mediating gene expressions. However, it is less clear how its binding is influenced by physiological stimuli and how its altered binding affects transactivator acetylation and binding. In this study, we determined p300 binding to a core cyclooxygenase-2 (COX-2) promoter region by chromatin immunoprecipitation and streptavidin-agarose pull-down assays in basal and tumor necrosis factor-alpha (TNFalpha)-treated human foreskin fibroblasts. We found basal binding of p300, p50/p65 NF-kappaB, cyclic AMP regulatory element-binding protein-2, CCAAT/enhancer-binding protein beta, and c-Jun. p50/p65 and p300 binding was selectively increased by TNFalpha. Immunoprecipitation confirmed direct interaction of p300 with NF-kappaB and the other involved transactivators. p50 acetylation was detected in resting cells and was increased by TNFalpha or lipopolysaccharide. Overexpression of p300 augmented p50 acetylation, which was attenuated by deletion of its histone acetyltransferase domain. Enhanced p50 acetylation correlated with increased p50 binding to COX-2 promoter and transcriptional activation. Co-transfection of E1A with p300 abrogated p50 acetylation and p50 binding. These findings suggest that up-regulation of p300 binding and its acetylation of NF-kappaB occupies a central position in COX-2 promoter activation.