In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, growth factors, and angiogenic factors. Since NF-kappaB is a key molecule involved in the regulation of several of these factors, here, we examined NF-kappaB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-kappaB was observed as early as 5 to 15 min postinfection by KSHV, and translocation of p65-NF-kappaB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IkappaB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-kappaB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-kappaB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-kappaB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription factors, including AP-1 family members, as well as several cytokines, growth factors, and angiogenic factors, which were significantly affected by NF-kappaB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-kappaB to regulate viral and host cell genes and thus possibly regulates the establishment of latent infection.