Nijmegen breakage syndrome (NBS), which in the past also has been classified as a variant of ataxia telangiectasia (AT), is characterized by cancer proneness and extreme sensitivity to ionizing radiation. We investigated the DNA damage responses of four independent primary NBS fibroblast cell lines. Following a low dose of ionizing radiation, p53 is mostly induced with slower kinetics and shows more transient induction in NBS fibroblasts. Nonetheless, this damage-induced protein appears biologically functional: unsynchronized and synchronized NBS cells show a G1 arrest after ionizing radiation as determined by bivariate flow cytometry. Neither an AT cell line nor a NBS cell line transformed with human papillomavirus genes E6 and E7 shows a G1 arrest. Furthermore, NBS cells show a normal G2 block, unlike that shown for AT cells. These data provide a cellular distinction between NBS and AT, thereby clearly separating the NBS from the AT syndrome.