The p300/cAMP response element-binding protein-binding protein (CBP) family members include human p300 and cAMP response element-binding protein-binding protein, which are both important transcriptional coactivators and histone acetyltransferases. Although the role of these enzymes in transcriptional regulation has been extensively documented, the molecular mechanisms of p300 and CBP histone acetyltransferase catalysis are poorly understood. Herein, we describe the first detailed kinetic characterization of p300 using full-length purified recombinant enzyme. These studies have employed peptide substrates to systematically examine the substrate specificity requirements and the kinetic mechanism of this enzyme. The importance of nearby positively charged residues in lysine targeting was demonstrated. The strict structural requirement of the lysine side chain was shown. The catalytic mechanism of p300 was shown to follow a ping-pong kinetic pathway and viscosity experiments revealed that product release and/or a conformational change were likely rate-limiting in catalysis. Detailed analysis of the p300 selective inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.