Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g. p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα activation by histone deacetylase inhibitors and p300. The conservation of the ERα a acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.