Checkpoint kinases Chk1 and Chk2 are two key components in the DNA damage-activated checkpoint signaling pathways. To distinguish the roles of Chk1 and Chk2 in S and G(2) checkpoints after DNA damage, derivatives of the human breast cancer cell line MDA-MB-231 were established that express short hairpin RNAs to selectively suppress Chk1 or Chk2 expression. DNA damage was induced with the topoisomerase I inhibitor SN38 which arrests cells in S or G(2) phase depending on concentration. Depletion of Chk1 resulted in loss of S phase arrest upon incubation with SN38, but the cells still arrested in G(2). Suppression of Chk2 had no impact on cell cycle arrest, while cells concurrently suppressed for both Chk1 and Chk2 still arrested primarily in G(2) suggesting the presence of an alternate checkpoint regulator. One critical target for Chk1 is Cdc25A which is phosphorylated and degraded to prevent cell cycle progression. Cells arrested in G(2) in the absence of Chk1/Chk2 still showed regulation of Cdc25A consistent with the action of an alternate kinase. One candidate for an alternate checkpoint kinase is MAPKAPK2 (MK2), yet this kinase was minimally activated by DNA damage and its inhibition did not facilitate either S or G(2) progression. Furthermore, we were unable to substantiate the recent observation that the Chk1 inhibitor UCN-01 inhibits MK2. These results show that Chk1, but neither Chk2 nor MK2, is an important regulator of S phase arrest, and suggest that an additional kinase can contribute to the G(2) arrest.