Although radiation-induced gene expression has been extensively studied, most of the studies to date have focused on that after single-dose irradiation. As split-dose irradiation, rather than single-dose irradiation, is usual in clinical situations, we investigated the effects of split-dose irradiation on nuclear factor kappaB (NF-kappaB) in the human rectum carcinoma cell line, LS174T. After either single- or split-dose irradiation with a different interval, nuclear localization of NF-kappaB was examined by Western blot and immunofluorescence and its DNA-binding activity was measured by ELISA-based assay. Irradiation-induced NF-kappaB nuclear accumulation and DNA binding activity increased in a dose-dependent manner. The peak of NF-kappaB nuclear accumulation and DNA binding activity was seen 2 to 6 hours after a single dose of 4 Gy irradiation and returned to control levels after 12 hours. In split-dose irradiation, NF-kappaB activity was similar after the first and second doses of 4 Gy irradiation separated by 12 hours. In addition, NF-kappaB activity was decreased by lengthening the interval between irradiation. The cell survival, which was assessed by colony formation assay, showed inverse correlation to this: the surviving fraction was higher after split-dose irradiation than after single-dose irradiation of the same total dose and it increased as the interval between irradiation was lengthened. Thus the present results showed a correlation between NF-kappaB activation and the repair of sublethal damage in split-dose irradiation.