Structure-activity relationships for inhibition of DNA-dependent protein kinase (DNA-PK) have been defined for substituted chromen-4-ones. For the 2-amino-substituted benzo[h]chromen-4-ones, a morpholine substituent at this position was essential for activity. Small libraries of 6- and 7-alkoxy-substituted chromen-4-ones showed that a number of 7-alkoxy-substituted chromenones displayed improved activity. Focused libraries incorporating 6-, 7-, and 8-aryl and heteroaryl substituents were prepared. In these cases, 6- and 7-substitution was disfavored, whereas 8-substitution was largely tolerated. Surprisingly, two compounds, 2-N-morpholino-8-dibenzofuranyl-chromen-4-one (NU7427, 32{38}) and the 2-N-morpholino-8-dibenzothiophenyl-chromen-4-one (NU7441, 32{26}) were excellent inhibitors (IC50 vs DNA-PK = 40 and 13 nM, respectively). The ring-saturated analogue 2-N-morpholino-8-(6',7',8',9'-tetrahydrodibenzothiophene)chromen-4-one, 36, retained potent activity (IC50 vs DNA-PK = 23 nM). The dibenzothiophene 32{38} sensitized HeLa cells to ionizing radiation in vitro, with dose modification factors of 2.5 at 10% survival being observed at 0.5 microM. The cytotoxicity of the topoisomerase II inhibitor etoposide was also potentiated.