Nijmegen breakage syndrome (NBS) patients and carriers are predisposed to malignancy and are often treated with X-irradiation. In the present study, the single-cell gel electrophoresis (Comet) assay was used to examine radiation-induced DNA damage and repair in peripheral blood mononuclear cells from NBS patients (n = 13) and carriers (n = 36) of six unrelated families. Cells from apparently healthy donors (n = 10) and from breast cancer patients with normal clinical radiosensitivity (n = 10) served as controls. Cells were irradiated with 5 Gy of X-rays and assayed for initial DNA damage and for residual DNA damage after 40 min of repair; the kinetics of DNA repair also was estimated. In addition, the nuclear area of unirradiated cells was extracted from the Comet data. The initial radiation-induced DNA fragmentation indicated that cells from members of two out of six NBS families were significantly more sensitive to X-irradiation than cells from the controls. Cells from four NBS families had longer DNA repair half-time values, while cells from five NBS families had significantly increased residual DNA damage following repair. The mean nuclear area of unirradiated cells processed in the Comet assay was 1.3-fold higher in cells from all NBS families than in the controls (P < 0.05). Notably, the Comet assay parameters (initial and residual DNA damage and the repair kinetics) of irradiated NBS cells predicted the carrier status of the majority (86%) of blindly tested individuals. The prediction of NBS status was higher if the nuclear area of unirradiated cells was used as the endpoint. The results of this study suggest that the impaired radiation response of NBS cells should be taken into account if radiotherapy of NBS patients and carriers is required.