It was found that Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-leucy]-L-lysyl-methyl ester (1) and Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-phenylalanyl]-L-lysyl-methyl ester (2) could cleave supercoiled DNA such as PUC19 efficiently in 40 mM Britton-Robinson buffer. The cleavage activities for both were investigated by agarose gel electrophoresis. The T4 ligase experiments implied that the cleavage of DNA occurs via a hydrolytic path. The results showed that the cleavage reaction of DNA is dependent on the value of pH and ionic strength in the solution. DNA cleavage is more efficient by N-phosphoryl branched peptide 2 than by N-phosphoryl branched peptide 1. The experiments also show that hydrolysis of DNA by N-phosphoryl branched peptide 1 was accelerated in the presence of Mg2+ or Zn2+ ions. The interactions of DNA with N-phosphoryl branched peptides were also characterized by melting temperature measurements and circular dichroism (CD) techniques. On the basis of experimental data, the possible mechanism of interactions between DNA with N-phosphoryl branched peptides was discussed.