Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit−/− cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit−/− cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression. © 2004 Wiley-Liss, Inc.