The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulations HAT activity. Here we have developed a strategy to introduce acetyl-Lys at Lip to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA Substrate analogue and that HAT activity correlates positively with degree of acetylation. This Study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.