Human diseases characterized by a high sensitivity to DNA interstrand cross-links (ICL) and predisposition to malignance include Nijmegen breakage syndrome (NBS) and Fanconi anemia (FA), which is further classified to three groups: (1) FA core-complex group; (2) FA-ID complex group; and (3) breast cancer (BRCA)-defective group. The relationships between these four groups and the basic defect in ICL repair remain unclear. To study the details of ICL repair in NBS and FA, a highly sensitive PPB (psoralen-polyethylene oxide-biotin) dot blot assay was developed to provide sensitive quantitative measurements of ICL during the removal process. Studies utilizing this assay demonstrated a decreased rate of ICL removal in cells belonging to the FA core-complex group (e.g. groups A and G) and FA-ID complex group (group D2), while ICL removal was restored to normal levels after these cells were complemented with wt-FANCA, wt-FANCG and wt-FANCD2. Conversely, FA-D1 cells with a defective BRCA2 protein displayed normal ICL removal, although they were compromised with respect to recombination. This normal ICL removal rate in recombination-deficient cells was confirmed by using XRCC3-defective Chinese hamster cells, which are similarly compromised with respect to recombination and are sensitive to mitomycin C. The present study also showed that cells from patients with Nijmegen breakage syndrome were defective in ICL removal, while they were impaired in the recombination. These results indicate an obvious defect of FA and NBS in the ICL repair process, except in the BRCA-defective group, and a separate step of recombination-mediated repair pathway between the BRCA group and NBS.