A series of DNA-intercalating potential antitumor agents, (amino)alkyl-substituted 2,3-dihydro-1H,7H-pyrimido[5,6, 1-de]acridine-1,3,7-triones, has been prepared by aminolysis of the corresponding 6-chloro derivative with a suitable omega-aminoalkylamine. The noncovalent DNA-binding properties of these compounds have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives toward eight tumor cell lines, including human colon adenocarcinoma (HT29, LoVo sensitive and LoVo/Dx (doxorubicin-resistant)) and human ovarian carcinoma (A2780 sensitive, A2780cisR (cisplatin-resistant), CH1, CH1cisR (cisplatin-resistant), and SKOV-3) cells, are described and compared to that of reference drugs. The cytotoxic activity often parallels the observed DNA affinities, for almost all the target compounds. Interesting structure-activity relationships have been found. The octanol/water partition coefficients have also been calculated, but there was no correlation either with cytotoxicity values or with resistance index. Three highly DNA-affinic analogues, 9 and 15f,15h, have been identified with a useful broad spectrum of cytotoxic activity.