Mammalian cells undergo cell cycle arrest in response to DNA damage due to the existence of multiple checkpoint response mechanisms. One such checkpoint pathway operating at the G1 phase is frequently lost in cancer cells due to mutation of the p53 tumor suppressor gene. However, cancer cells often arrest at the G2 phase upon DNA damage, due to activation of another checkpoint pathway that prevents the activation Cdc2 kinase. The kinases, Chk1, Wee1, and Myt1 are key regulators of this G2 checkpoint, which act directly or indirectly to inhibit Cdc2 activity. Here we show that RNA interference (RNAi)-mediated down-regulation of Wee1 kinase abrogated an Adriamycin“-induced G2 checkpoint in human cervical carcinoma Hela cells that are defective in G1 checkpoint response. Wee1 down-regulation sensitized HeLa cells to Adriamycin“-induced apoptosis. Down-regulation of Chk1 kinase in Hela cells also caused a significant amount of cell death independent of DNA damage. In contrast, Myt1 down-regulation also abrogated Adriamycin“-induced G2 arrest but did not cause substantial apoptosis. Reduction in Wee1, Chk1, or Myt1 levels did not sensitize normal human mammary epithelial cells (HMEC) cells to Adriamycin“-induced apoptosis unlike the situation in Hela cells. Our study reveals distinct roles for Chk1, Wee1, and Myt1 in G2 checkpoint regulation. The data reported here support the attractiveness of Wee1 and Chk1 is as molecular targets for abrogating the G2 DNA damage checkpoint arrest, a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.