The investigational anticancer agent 7-hydroxystaurosporine (UCN-01)abrogates the G₂ checkpoint in tumor cells and sensitizes them to the lethal effects of genotoxic anticancer agents. On the basis of the role of the Cdc25C phosphatase in maintenance of this damage-inducible checkpoint, we hypothesized that UCN-01 inhibits a component of the signal transduction pathway that modulates Cdc25C phosphorylation. Of the three kinases known to phosphorylate Cdc25C on Ser²¹⁶, both checkpoint kinase 1 (hChk1) and Cdc25C-associated protein kinase 1 (cTAK1) were potently inhibited by UCN-01 with IC₅₀s of 11 and 27 nm,respectively. Treatment of K562 erythroblastoid leukemia cells with similar drug concentrations resulted in decreased levels of Ser²¹⁶ phosphorylation of Cdc25C and complete disruption of the γ-radiation-induced G₂ checkpoint. In contrast to hChk1, the hChk2 kinase was 100-fold more resistant to inhibition by UCN-01 (IC₅₀, 1040 nm). These results suggest that disruption of the DNA damage-induced G₂ checkpoint by UCN-01 is mediated through the inhibition of the Cdc25C kinases, hChk1 and cTAK1, and that hChk2 activity is not sufficient to enforce the G₂ checkpoint in cells treated with a pharmacological inhibitor of hChk1.