To elucidate the possible effect of NFκB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFκB constitutive inhibitor, mlκB(Saos2-mlκB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mlκB cells but not in Saos2-EV control cells, with intact NFκB activity. We find as expected, that this NFκB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFκB signaling in Saos2-mlκB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFκB resulted in decreased expression of the DNA damage protein GADD45β, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mlκB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-cfrom mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mlκB cells. Antisense inhibition of GADD45κ in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFκB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.