The recruitment of the bromodomains of CREB-binding protein (CBP) and p300 by the acetylated myogenic transcription factor MyoD was previously shown to be critical for the enhanced MyoD transcriptional activity following acetylation at its Lys(99) and Lys(102) positions. However, the modes of binding interactions of the bromodomains of CBP and p300 with acetylated MyoD have not been well-characterized. In the current study, by employing a panel of MyoD peptides encompassing the 99 and 102 positions, we showed that Lys99 monoacetylation and Lys(99)/Lys(102) double acetylation defined the critical binding interfaces with the bromodomains of CBP and p300, respectively. This also represented the first identification of a recognition motif for the p300 bromodomain and revelation of the differential recognition motifs for the bromodomains of CBP and p300. This information could be exploited for developing novel tools for structural and functional studies of the highly homologous CBP and p300 transcriptional coactivators. (C) 2008 Elsevier Inc. All rights reserved.