To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B-2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B-2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B-2 to duplex DNA could advance its design as an antitumor agent.