Certain indeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antiproliferation, DNA binding affinity, and topoisomerases (topo I and topo II) inhibitory activities. The preliminary results are the following: (1) substituent of the aminoalkoxyimino side chain at C11 is important for antiproliferative activities in which the terminal amine preferred to be a tertiary or the cyclic five-membered pyrrolidino ring; (2) among the indeno[1,2-c]quinoline derivatives evaluated, (E)-6-hydroxy-9-methoxy-11H-indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (8c) was found to be one of the most cytotoxic agents with a GI50 value of 0.84, 0.89, and 0.79 microM against SAS, A549, and BT483, respectively, which is more active than camptothecin; (3) substituent at C6 is crucial for the selective cytotoxicity in which the OH group is the most preferred while hydrogen or piperazine exhibited cytotoxicity on both cancer cells and Detroit-551; (4) a positive correlation of antiproliferative activity, DNA binding affinity, and topo I and topo II inhibitory activities has been observed for indeno[1,2-c]quinoline derivatives; (5) compound 8c induced DNA fragmentation may through caspase-3 activation, phosphorylation of the histone protein H2AX at Ser139 (gamma-H2AX), and PARP cleavage; (6) compound 8c demonstrated significant tumor regression in the human breast xenograft model; (7) indeno[1,2-c]quinoline derivatives are a new class of molecules that have the potential to be developed as dual topo I and topo II inhibitory agents.