Mammalian Chk1 and Chk2 protein kinases are two important components of the G2DNA damage checkpoint. They are activated by upstream kinases (ataxia telangectasiamutated gene and ATM and Rad 3 related gene) and interfere with the activity of thecdc2/cyclinB1 complex, necessary for the G2-M transition, through the inactivation of thecdc25 phosphatases (cdc25A and cdc25C). To understand the role of Chk1 and Chk2 in thecellular response to different anticancer agents, we knocked down the expression of eachprotein or simultaneously of both proteins by using the small interfering RNA techniquein the HCT-116 colon carcinoma cell line and in its isogenic systems in which p53 and p21have been inactivated by targeted homologous recombination. We here show thatinhibition of Chk1 but not of Chk2 in p21-/- and p53-/- cells caused a greater abrogationof G2 block induced by ionizing radiation and cis-diamine-dichloroplatinum treatmentsand a greater sensitisation to the same treatments than in the parental cell line with p53and p21 wild type proteins. These data further emphasise the role of Chk1 as a moleculartarget to inhibit in tumors with a defect in the G1 checkpoint with the aim of increasingthe selectivity and specificity of anticancer drug treatments.