Cell cycle checkpoint proteins play critical roles in maintaining genomic stability and integrity to prevent the development of cancer and hereditary diseases. Here we report the isolation of a novel mouse gene encoding the protein MmRad24 [MmRad24 is the mouse homologue of HRad17, which was described recently by A. E. Parker et al. (J. Biol. Chem., 273: 18340-18346, 1998)], which shares significant sequence and structural homology with the budding yeast Rad24 and its fission yeast counterpart Rad17, both of which are required for DNA damage checkpoints. Confocal microscopy revealed that the green fluorescent protein-tagged MmRad24 protein is localized to the nucleus in living cells. Fluorescence-activated cell-sorting analysis showed that overexpression of the wild-type MmRad24 in diploid fibroblast WI-38 cells caused a significant G2 arrest of the cell cycle, whereas overexpression of a mutant MmRad24 (mutated on the nucleotide-binding site) that likely functions as a dominant-negative protein resulted in a defect in cell cycle arrest after DNA damage treatment as measured by bromodeoxyuridine pulse-chase labeling experiments. Taken together, these results suggest that the mammalian Rad24 protein may function as a critical gatekeeper in DNA damage checkpoint control.