Evidence suggests that genome chromatinization and the posttranslational modification of histones are involved in the regulation of viral gene expression, including the human cytomegalovirus (HCMV). We performed a ChIP-on-Chip assay to determine whether histone deacetylases (HDACs) interact with HCMV genomic DNA on a global level. Surprisingly, we found that HDAC3, but not HDAC2, interacts not only with the major immediate early (MIE) promoter but also with the entire MIE locus, suggesting a heterogeneous interaction of HDAC3 with HCMV DNA. The interaction of HDAC3 with the MIE region is related to inhibition of viral replication because HDAC3 inhibitors enhanced HCMV replication.