Freshly isolated macrophages from mature mice are poorly or nonpermissive for infections with HSV. However, despite lack of significant viral replication, HSV infection has been demonstrated to induce substantial cell death among macrophages. To determine if HSV-induced cytotoxicity of macrophages is due to apoptosis, peritoneal macrophages were obtained from C57BL/6 (B6) mice, and apoptosis was analyzed following HSV-2 infection in vitro. Macrophages underwent apoptosis upon HSV-2 infection indicated by annexin V staining, labelling of DNA strand breaks and electronmicroscopy. Apoptosis was associated with macrophage activation demonstrated by upregulation of MHC class II and Mac-1 surface expression. Though there was also an upregulation of Fas (Apo-1/CD95) and tumor necrosis factor (TNF)- receptor 1 (TNF-R1) pathways, inhibition of Fas by soluble Fas and blocking of TNF-α using a TNF-binding protein did not prevent HSV-induced apoptosis. Moreover, apoptosis was not impaired in HSV-2 infected macrophages from Fas- deficient B6-lpr/lpr mice suggesting involvement of other apoptosis pathways, or activation of Fas or TNF-R pathways downstream of the receptor level. The present results demonstrate that HSV-2 infection leads to activation and subsequent apoptosis in peritoneal macrophages independent of Fas or TNF-R1 signaling.