Several putative NF-kappa B-binding sites in the ICP0 and Vmw65 herpes simplex virus type-1 (HSV-1) genes have been identified. Oligonucleotides encoding some of these sites bind specifically to purified NF-kappa B protein and an NF-kappa B-like protein in nuclear extracts of phorbol ester- or cycloheximide-induced human embryonic lung (HEL) cells. HSV-1 infection of HEL cells induced a nuclear factor that binds specifically to kappa B sites in the ICP0 and Vmw65 gene regions and comigrates with complexes formed by purified NF-kappa B. The HSV-1-inducible nuclear factor bound to the authentic immunoglobulin (Ig) kappa B site. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing two copies of the Ig kappa B site upstream of the c-fos promoter (kappa B2-CAT) showed activity in HEL cells. HSV-1 infection of kappa B2-CAT-transfected HEL cells, however, induced a dramatic increase in CAT activity; mutation in the NF-kappa B-binding site of kappa B2-CAT abolished the inducibility of CAT gene expression. Our results demonstrate that the HSV-1 ICP0 and Vmw65 gene regions contain binding sites for NF-kappa B, and that HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.