We have previously demonstrated that wild-type herpes simplex virus type 1 (HSV-1), as well as nonreplicating UV-inactivated HSV-1, promptly activates the nuclear factor-kappaB (NF-kappaB) in U937 monocytoid cells and that glycoprotein D (gD) of HSV-1 is sufficient by itself to exert a similar effect. We then investigated the signaling pathway used by HSV-1 to initiate NF-kappaB activation and, particularly, whether our observation could be related to the capability of HSV-1-gD to directly stimulate NF-kappaB through its interaction with the herpes virus entry receptor A (HveA). Here we report that: (a) co-cultivation of U937 cells with an adherent cell line expressing wild-type gD on its surface led to increased NF-kappaB activation, while co-cultivation with the same adherent cell line expressing a mutated form of gD, lacking the capability to bind HveA, did not cause the same effect; (b) exposure to UV-inactivated HSV-1 induced the activation of NF-kappaB in HveA-expressing U937 and THP-1 cells, but not in non-HveA-expressing HEp-2 cells; and (c) activation of NF-kappaB in U937 and THP-1 cells exposed to soluble gD was inhibited by an antibody able to interfere with gD-HveA interaction. These results suggest that HSV-1-gD-HveA interaction initiates a signal transduction pathway leading to NF-kappaB activation.