During lytic infection by herpes simplex virus type 1 (HSV-1), histones are present at relatively low levels on the viral genome. However, the mechanisms that account for such low levels - how histone deposition on the viral genome is blocked or how histones are removed from the genome - are not yet defined. In this study, we show that histone occupancy on the viral genome gradually increased with time when transcription of the viral immediate-early (IE) genes was inhibited either by deletion of the VP16 activation domain or by chemical inhibition of RNA polymerase II (RNAP II). Inhibition of IE protein synthesis by cycloheximide did not affect histone occupancy on most IE promoters and coding regions but did cause an increase at delayed-early and late gene promoters. IE gene transcription from HSV-1 genomes associated with high levels of histones was stimulated by superinfection with HSV-2 without altering histone occupancy or covalent histone modifications at IE gene promoters. Moreover, RNAP II and histones cooccupied the viral genome in this context, indicating that RNAP II does not preferentially associate with viral genomes that are devoid of histones. These results suggest that during lytic infection, VP16, RNAP II, and IE proteins may all contribute to the low levels of histones on the viral genome, and yet the dearth of histones is neither a prerequisite for nor a necessary result of VP16-dependent transcription of nucleosomal viral genomes.