The process of transcriptional activation directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) was investigated by in vivo footprinting using ligation-mediated polymerase chain reaction in a human epithelial cell line infected with human herpes simplex virus type 1 (HSV-1) or human herpes virus 6 (HHV-6). Infection with both viruses induces a remarkable enhancement in LTR-mediated gene expression that correlates with a change in the pattern of protein binding to the downstream kappa B site of the enhancer region. In HHV-6 infected cells, this change in the genomic footprinting pattern is concomitant with the induction of specific enhancer-binding proteins in the nucleus. The similarity of these events to those detected in other previously investigated experimental systems suggests that the LTR enhancer region is the ultimate target for the induction of the HIV-1 transcriptional response upon stimuli acting through different upstream pathways.