Rad9, Rad1 and Hus1 are critical for the cell cycle checkpoint and can form a heterotrimer complex called 9-1-1 complex which was supposed to play important roles in the cell cycle checkpoint and other activities required for the maintenance of genome integrity. However, lack of high quality anti-Rad1 antibodies has seriously hindered the research on Rad1 as well as working mechanisms of the 9-1-1 complex at molecular level. In this study, a mouse anti-Rad1 monoclonal antibody (mAb) was successfully generated. The mAb possesses high affinity and specificity, and recognizes both endogenous mouse Rad1 (mRad1) and human Rad1 (hRad1) proteins and was successfully used in ELISA, Western blot analysis, immunoprecipitation and immunofluorescence assays. Using this mAb, we found that mRad1 protein expression was increased in Rad9^+/+ mouse embryonic stem (MES) cells after hydroxyurea (HU, a genotoxic agent) treatment while not in Rad9^-/- MES cells, suggesting that mRad1 expression is under Rad9 regulation. Furthermore, endogenous mRad1 was distributed mainly in the cytoplasm and did not migrate to the nucleus after HU treatment, contradicting the generally accepted hypothesis that Rad9, Rad1 and Hus1 form the 9-1-1 complex in the nucleus in response to genotoxic stresses. In summary, the exact molecular roles of Rad1 and the 9-1-1 complex are likely more complicated than previously expected and this anti-Rad1 mAb is a powerful tool for the future investigation on Rad1 as well as the 9-1-1 complex.