Human cytomegalovirus DNA is packaged in virions without histones but associates with histones upon reaching the nucleus of an infected cell. Since transcription is modulated by the interplay of histone modifications, we used chromatin immunoprecipitation to detect acetylation and methylation of histone H3 at viral promoters at different times during the viral replication cycle. Histone H3 at immediate-early promoters is acetylated at the start of infection, while it is initially methylated at early and late promoters. Acetylation at immediate-early promoters is dynamic, with a high level of activating modifications at 3 and 6 h postinfection (hpi), followed by a marked reduction at 12 hpi. All viral promoters, as well as nonpromoter regions, are modified with activating acetylations at 24 to 72 hpi. The transient reduction in histone H3 acetylation at the major immediate-early promoter depends on the cis-repressive sequence to which the UL122-coded IE2 protein binds. A mutant virus lacking this element exhibited decreased IE2 binding at the major immediate-early promoter and failed to show reduced acetylation of histone H3 residing at this promoter at 12 hpi. Our results demonstrate that cytomegalovirus chromatin undergoes dynamic, promoter-specific histone modifications early in the infectious cycle, after which the entire chromosome becomes highly acetylated.