Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-kappaB and downstream cellular genes. To better understand the mechanism of NF-kappaB activation by reovirus, NF-kappaB signaling intermediates under reovirus control were investigated at the level of Rel, IkappaB, and IkappaB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-kappaB inhibitor IkappaBalpha but not the structurally related inhibitors IkappaBbeta or IkappaBepsilon. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKalpha but not IKKbeta is required for reovirus-induced NF-kappaB activation and apoptosis. Despite the preferential usage of IKKalpha, both NF-kappaB activation and apoptosis were attenuated in cells lacking IKKgamma/Nemo, an essential regulatory subunit of IKKbeta. Moreover, deletion of the gene encoding NF-kappaB-inducing kinase, which is known to modulate IKKalpha function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-kappaB/Rel activation involving IKKalpha and IKKgamma/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells.