Herpes simplex virus (HSV) is a large DNA virus which is characterized by its ability to form latent infections in neurons of the peripheral nervous system. Although histones are found in the capsids of small DNA viruses (papovaviruses), none are found in the capsids of large HSV. However, after entry into the infected cell nucleus, the HSV genome begins to associate with nucleosomes during the earliest stages of infection. In contrast, late during infection, newly replicated viral DNA does not appear to associate with nucleosomes, suggesting that histones are deposited specifically on input viral DNA. The mechanisms of deposition and removing histones from the viral genome are unclear. Recently, histone chaperones, involved in the assembly and disassembly of nucleosomes, have been identified. Human antisilencing factor 1 (Asf1) is one such factor which is involved in both the assembly and disassembly of nucleosomes in cellular systems. In this study, we have examined the effect of small interfering RNA (siRNA) knockdown of Asf1a on HSV infections in HeLa cells. Both viral replication and growth were found to be decreased. Also, viral DNA was significantly less protected from micrococcal nuclease (MNase) digestion up to 6 h postinfection (hpi). However, transcription of the immediate early (IE) genes ICP0 and ICP4 was significantly upregulated at 3 h postinfection. Also, these genes were found to be less protected from MNase digestion and, therefore, less associated with nucleosomes. These results suggest that Asf1a plays a role in regulating IE genes by assembling chromatin onto histone-free viral DNA by 3 h postinfection.