Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polalpha and not Polepsilon or Poldelta by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polalpha and Chk1 causes an accumulation of gamma-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polalpha with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polalpha can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polalpha-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polalpha complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polalpha as an important component of the signal transduction cascade that activates the intra-S checkpoint.