The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor kappaB (NF-kappaB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-kappaB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-kappaB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-kappaB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-kappaB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-kappaB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-kappaB activation by HSV-1 gD.