A series of pyrenyl-macrocyclic polyamines 5a-5c have been prepared and characterized. Their DNA-cleavage properties were examined under physiological conditions. Without the presence of other additives, the DNA cleavage ability of 5a-5c showed the order of 5c>5a>5b. Absorption and fluorescence experiments showed the binding affinity of 5a-5c to DNA. The interactions of 5a-5c with CT-DNA indicated that the DNA binding ability followed an order according to their cleavage efficiency. All the results indicated that the structures of amino-acid bridge in the ligands may affect the DNA binding and cleavage ability. The cleavage-mechanism studies indicated that singlet oxygen and superoxide free radicals were involved in the catalytic DNA cleavage process.