A tumorigenic role of the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks (DSBs) has been suggested by the finding of a significant association between increased breast cancer risk and a cooperative effect of single nucleotide polymorphisms (SNPs) in NHEJ genes. However, the lack of an association between hereditary breast cancer and defective NHEJ genes prevents conclusions from being drawn about a link between NHEJ and breast cancer development. Recently, BRCA1-deficient mouse embryonic fibroblasts were found to have significantly reduced NHEJ activity, suggesting an accessory role of BRCA1 in NHEJ. The present study was performed to confirm this observation in human breast cancer cell lines and to examine whether the interaction between BRCA1 and NHEJ was of tumorigenic significance. Support for this hypothesis came from the findings that (a) a case-control study (469 breast cancer patients and 740 healthy controls) showed that the breast cancer risk associated with high-risk genotypes of NHEJ genes was significantly modified by the BRCA1 genotype. A significant increase in the cancer risk associated either with harboring one additional putative high-risk NHEJ genotype or with the joint effect of having reproductive risk factors (reflected by an interval of > or =12 years between menarche and first full-term pregnancy) and a higher number of high-risk genotypes of the NHEJ genes was only seen in women with at least one variant BRCA1 allele (i.e., the Glu/Gly or Gly/Gly forms of BRCA1 Glu(1038)Gly); and (b) a phenotype-based study measuring in vitro and in vivo NHEJ capacity showed that the precise end-joining capacity was different in breast cancer cell lines with different BRCA1 statuses being higher in BRCA1-expressing MCF-7 cells than in HCC1937 cells (defective BRCA1 expression). Furthermore, this end-joining capacity was decreased in MCF-7 cells in which BRCA1 expression was blocked using small interfering RNA and increased in HCC1937 transfected with full-length BRCA1. Because BRCA1 is a well-documented breast cancer susceptibility gene, this association between NHEJ and BRCA1 not only suggests a role of BRCA1 in NHEJ but also provides essential support for the tumorigenic contribution of NHEJ in breast cancer development.