Efficient clearance of virus infections depends on the nature of the host response raised by the infected organism. A proinflammatory cell-mediated immune response is important for elimination of many viruses, including herpesviruses. Macrophages are intimately involved in generation of a proinflammatory response, the initiation of which involves activation of the transcription factor NF-kappaB. However, the mechanisms of HSV-induced NF-kappaB activation are poorly understood. In this study we demonstrate that activation of NF-kappaB by HSV in macrophages is dependent on a functional viral genome and proceeds through a mechanism involving the cellular IkappaB kinase, as well as the upstream kinases TGF-beta-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-kappaB-inducing kinase. Furthermore, we show that HSV triggers NF-kappaB activation by a signaling pathway involving oxidative stress in mitochondria and intracellular calcium, because specific inhibition of mitochondria-derived reactive oxygen intermediates, as well as mitochondrial calcium channels, prevented NF-kappaB activation. Together, these results point to mitochondria as cellular checkpoints able to initiate NF-kappaB activation after virus infection and also show that the cellular NF-kappaB-regulating kinases IkappaB kinase, TGF-beta-activated kinase 1, mitogen-activated kinase/extracellular signal-regulated kinase kinase 1, and possibly NF-kappaB-inducing kinase, are essential components in the HSV-induced signaling pathway.