Structural abnormalities of chromosomes, including translocations and deletions, are extremely frequent in human cancer cells and particularly in breast cancer cells. One hypothesis to account for these alterations is a deficiency in the repair of DNA double-strand breaks (DSB). This repair process relies on two distinct pathways, homologous recombination (HR) and non-homologous DNA end joining (NHEJ). To investigate this latter pathway, we have studied the ability of cell-free extracts from a variety of human cells to rejoin different types of DSBs. The end joining activity of eleven sporadic breast cancer cell lines (BCCLs) was compared with that of control cells including primary human fibroblasts and cells harbouring a limited number of chromosome abnormalities. In vitro rejoining activity was not detected in extracts from MO59J DNA-PKcs-deficient cells and was strongly inhibited by wortmannin in control extracts. In contrast, most sporadic BCCLs and BRCA1 or BRCA2 deficient cells demonstrated similar efficiencies and accuracies of in vitro NHEJ than control cells. Only two BCCLs, SKBR3 and MDA-MB-453 exhibited decreased in vitro NHEJ. This study therefore indicates that a major defect in the NHEJ pathway is unlikely to account for the high number of chromosomes abnormalities observed in sporadic and hereditary BCCLs.