To determine whether p300 is involved in inducible NO synthase (iNOS) transcriptional regulation, we evaluated the effect of p300 overexpression on iNOS expression and characterized p300 binding to iNOS promoter in RAW 264.7 cells. p300 overexpression increased iNOS expression which was abrogated by deletion of the histone acetyltransferase (HAT) domain (Delta1472-1522). DNA-binding and chromatin immunoprecipitation assays revealed binding of p300 to several DNA-bound transactivators at basal state. Following stimulation with LPS plus IFN-gamma, binding of p300, p50/p65 NF-kappaB, and IFN-regulatory factor-1 was increased by approximately 2-fold. Nuclear p50 was complexed with and acetylated by p300 at the basal binding state which was increased by LPS and IFN-gamma stimulation. p300 overexpression resulted in increased p50 acetylation which was reduced by HAT mutation. p50 acetylation correlated with increased NF-kappaB binding and enhanced p300 recruitment. Co-overexpression of E1A abolished the augmentation of p50 acetylation and p50 binding induced by p300 overexpression, and a correlative suppression of p300 recruitment to the complex. We conclude that p300 is essential for iNOS transcription. Our results suggest that p300 HAT acetylates the p50 subunit of NF-kappaB, thereby increasing NF-kappaB binding and NF-kappaB mediated transactivation.