The sequence-specificity, and DNA alkylating activity of the conjugate 1, which consists of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with indole linker, were investigated in the absence or presence of partner Py-Im polyamide 2. High-resolution denaturing polyacrylamide gel electrophoresis showed that the specificity of DNA alkylation by 1 modulated in the presence of partner 2. We found that sequence-specific DNA alkylation by 1 and 2 with 10 base pair (bp) match recognition sequence through heterodimer formation. This result indicates one possibility of DNA alkylation with longer recognition sequence by different two molecules.