Using a variety of biochemical and cell-based approaches, we show that estrogen receptor alpha(ER alpha) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ER alpha ( Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ER alpha acetylated at Lys266 and Lys268. The acetylation of ER alpha by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/ nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ER alpha in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand- dependent activity of ER alpha in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand- dependent gene regulatory activity of ER alpha. Such regulation is likely to play a role in estrogen- dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.