Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM ( ataxia telangiectasia, mutated)- and ATR ( ATM and Rad3-related)-defective cells, indicating a complementary role for these DNA-damageresponsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 ( Chk1) by RNA- mediated interference or drug treatment suppressed DNA- damage- induced centrosome amplification. Radiation- induced centrosome amplification was abrogated in Chk1(-/-) DT40 cells, but occurred at normal levels in Chk1(-/-) cells transgenically expressing Chk1. Expression of kinase- dead Chk1, or Chk1S345A, through which the phosphatidylinositol- 3- kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.