Fanconi anemia (FA) is a genetic disorder defined by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). MMC causes increased FA cell death, chromosome breakage, and accumulation in the G2 phase of the cell cycle. Recently, Fanconi anemia complementation group C (fac) gene knock-out mice have been developed, and SV40-transformed fibroblasts were established from fac homozygous knock-out (-/-), heterozygous (+/-), and wild-type mice (+/+). MMC sensitivity of these cell lines was assessed by three methods: colony-formation assay in the presence of MMC, chromosome breakage, and cell cycle analysis to detect G2 phase arrest. The fac knock-out fibroblasts (-/-) showed a significantly higher sensitivity to MMC than did fibroblasts from wild-type (+/+) or heterozygous (+/-) mice (three experiments). In addition, we analyzed hematopoietic progenitor colony assays of bone marrow cells from fac knock-out (-/-) and heterozygous (+/-) mice. CFU-E, BFU-E, and CFU-GM colony formation from fac nullizygous mouse progenitors was markedly diminished by MMC when compared to growth of progenitors from heterozygous mice. These results show that fac knock-out mouse cells mimic the behavior of human FA-C patient cells in terms of MMC hypersensitivity. The fac knock-out mouse may be used to model some aspects of human FA and should be useful for understanding the function of the FAC protein.