The human p 300/C BP‐a ssociating f actor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 Å crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N‐and C‐terminal protein segments. A correlation of the structure with the extensive mutagenesis data for PCAF and the homologous yeast GCN5 protein implicates the cleft and the N‐and C‐terminal protein segments as playing an important role in histone substrate binding, and a glutamate residue in the protein core as playing an essential catalytic role. A structural comparison with the coenzyme‐bound forms of the related N‐acetyltransferases, HAT1 (yeast histone acetyltransferase 1) and SmAAT (Serratia marcescens aminoglycoside 3‐N‐acetyltransferase), suggests the mode of substrate binding and catalysis by these enzymes and establishes a paradigm for understanding the structure–function relationships of other enzymes that acetylate histones and transcriptional regulators to promote activated transcription.