We report that a host nuclear protein of approximately 100 kDa binds to the tandemly reiterated DR2 sequence of herpes simplex virus type 1 (HSV-1). The DR2 sequence is a repeated component in the "a" sequence, which defines the signals for cleavage and encapsidation of viral DNA; the "a" sequence also contains the promoter regulatory signals for the gene encoding the viral neurovirulence factor, ICP34.5. Characterization of the host binding protein by means of gel shifts and DNase I footprinting revealed this protein is the eukaryotic transcription factor, Sp1. Furthermore, as judged from the sequence homology, the DR2 region contains clustered matches to the consensus binding site for Sp1. Comparison of the host factor and purified Sp1 (by means of gel shifts and footprinting) confirmed these findings. Since clustered DNA recognition elements represent unusually high affinity binding sites, these repeated Sp1 motifs proximal to the ICP34.5 gene suggest that this region may be a major Sp1 binding site in the viral genome.