Cyclin-dependent kinases (Cdk) promote cell proliferation, are often deregulated in human cancers, and are targets of ongoing cancer chemotherapy trials. We show here that Cdk activity is also required in human cells to maintain function of the Chk1 pathway, a key component of the response to DNA damage or stalled replication. Chk1 expression was markedly reduced in primary fibroblasts and U2OS osteogenic sarcoma cells by treatment with small molecule Cdk inhibitors or induction of a dominant-negative mutant of Cdk2. The findings of decreased Chk1 activity and accumulation of Cdc25A, a protein targeted for degradation by Chk1, confirmed that Chk1 function was impaired. Furthermore, Cdk inhibition triggered a DNA damage response, characterized by the accumulation of activated forms of ATM and Chk2 as well as nuclear foci containing phosphorylated substrates of ATM/ATR, including histone H2AX (gammaH2AX). Time course experiments showed that the bulk of ATM activation followed Chk1 down-regulation. Chk1 RNA interference combined with partial inhibition of DNA replication was sufficient to evoke the DNA damage response. Conversely, ectopic expression of Chk1 blunted induction of gammaH2AX foci by Cdk inhibitors, indicating that Chk1 down-regulation was necessary to elicit the full phenotype. Finally, both Cdk and Chk1 inhibitors enhanced the cytotoxity of etoposide, a DNA-damaging agent. These results define a pathway through which Cdk inhibition can mediate DNA damage and potentially enhance the efficacy of extant cancer chemotherapies.