The ability of herpes simplex virus type 1 (HSV-1) to activate NF-κB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-κB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IκBα and IκBβ. In this study, we identified loss of IκBα as a marker of NF-κB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IκBα loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IκBα loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-κB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-κB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IκBα and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-κB activation during HSV infection, highlighting the important role that NF-κB may play in infection.