NF-kappaB comprises a family of transcription factors that regulate key immune processes. In this study, the effects of orthopoxvirus infection upon the activation of NF-kappaB were examined. During the early phase of infection, cowpox virus can inhibit the induction of NF-kappaB-regulated gene expression by interfering with the process of IkappaBalpha degradation. Although either okadaic acid or tumor necrosis factor (TNF) treatment of infected cells can induce IkappaBalpha phosphorylation, further processing of IkappaBalpha is inhibited. These results suggest that cowpox virus is capable of inhibiting the activation of NF-kappaB at a point where multiple signal transduction pathways converge. Other orthopoxviruses affect NF-kappaB activity, but in a type-specific manner. Raccoonpox virus and vaccinia virus (Copenhagen strain) negatively affect NF-kappaB induction by TNF. In contrast, the modified vaccinia virus Ankara strain induces NF-kappaB activation, even in the absence of other stimuli. These findings suggest that orthopoxviruses may affect a broad range of virus-host interactions through their effects upon NF-kappaB activation. Moreover, because of the central role for NF-kappaB in immune processes and disease, these type-specific effects may contribute significantly to the immunogenic and pathogenic properties of poxviruses.