The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 microM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against Cdc25B(2) as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, Cdc25B(2), and Cdc25C with K(i) values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against Cdc25B(2) in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential mapping suggested the need for an electron-deficient 7-position for maximal inhibitor activity. Using a chemical complementation assay, we found that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.