Although histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here we describe an efficient single-step method to specifically purify histones located around an origin of replication from Saccharomyces cerevisiae. Using high-resolution MS, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that acetylation of histone H3 and H4 is dynamically regulated around an origin of replication, at the level of multiply acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S phase.