Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma. Activation of the cellular transcription factor nuclear factor-kappa B (NF-kappaB) is essential for latent persistence of HHV-8, survival of HHV-8-infected cells, and disease progression. We used reverse-transfected cell microarrays (RTCM) as an unbiased systems biology approach to systematically analyze the effects of HHV-8 genes on the NF-kappaB signaling pathway. All HHV-8 genes individually (n = 86) and, additionally, all K and latent genes in pairwise combinations (n = 231) were investigated. Statistical analyses of more than 14,000 transfections identified ORF75 as a novel and confirmed K13 as a known HHV-8 activator of NF-kappaB. K13 and ORF75 showed cooperative NF-kappaB activation. Small interfering RNA-mediated knockdown of ORF75 expression demonstrated that this gene contributes significantly to NF-kappaB activation in HHV-8-infected cells. Furthermore, our approach confirmed K10.5 as an NF-kappaB inhibitor and newly identified K1 as an inhibitor of both K13- and ORF75-mediated NF-kappaB activation. All results obtained with RTCM were confirmed with classical transfection experiments. Our work describes the first successful application of RTCM for the systematic analysis of pathofunctions of genes of an infectious agent. With this approach, ORF75 and K1 were identified as novel HHV-8 regulatory molecules on the NF-kappaB signal transduction pathway. The genes identified may be involved in fine-tuning of the balance between latency and lytic replication, since this depends critically on the state of NF-kappaB activity.