AbstractCoactivators p300 and CREB (cyclic adenosine monophosphate [cAMP]–response element binding protein)–binding protein (CBP) serve as an integrator for gene transcription. Their relative involvement in regulating cyclooxygenase-2 (COX-2) promoter activity had not been characterized. Using fibroblast and macrophage COX-2 transcription as a model, we determined p300 and CBP levels in nuclear extracts and their binding to a COX-2 promoter probe. CBP level was barely detectable and there was little CBP binding. In contrast, p300 was detectable in nucleus and its binding to a COX-2 promoter probe was enhanced by phorbol 12-myristate 13-acetate (PMA), interleukin-1β (IL-1β), or lipopolysaccharide (LPS). Binding of p300/CBP-associated factor (PCAF) was also up-regulated. COX-2 proteins and promoter activities induced by these agonists were augmented by p300 overexpression. Early region 1A (E1A), but not its deletion mutant, abrogated COX-2 expression induced by inflammatory mediators and with or without p300 overexpression. Molecular analysis of p300 revealed the requirement of multiple domains, including histone acetyltransferase (HAT) for COX-2 transactivation. Furthermore, roscovitine, an indirect inhibitor of p300 HAT, and histone deacetylase-1 transfection completely abolished COX-2 promoter activity. We conclude that p300 is the predominant coactivator that is essential for COX-2 transcriptional activation by proinflammatory mediators.