The synthesis of 9,9-difluoro-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (F2CBI), a difluorocyclopropane analog of the CC-1065 and duocarmycin alkylation subunits which represents the first such agent containing substitution of the reactive center in the natural products, is detailed. The core structure of F2CBI was prepared by an intramolecular metal carbenoid insertion reaction into a 1,1-difluoroalkene (74%) employing a p-quinonediazide, and its incorporation into F2CBI-TMI (24) provided a key analog of the duocarmycins. A study of the solvolysis of N-BOC-F2CBI (19) revealed that introduction of the difluorocyclopropane substitution increased the reactivity 500x without altering the inherent regioselectivity which occurred with nucleophilic addition to the difluoro substituted C9 cyclopropane carbon. A single-crystal X-ray structure analysis of 17 and its comparison with the X-ray structures of CBI and related agents beautifully reveal the structural origin of the difluoro substitution effects on the reactivity and regioselectivity of the cyclopropane cleavage reaction, The cyclopropane C-CF2-C bond angle is expanded, and the carbon-carbon bond opposite the difluoro substitution is lengthened to accommodate the preferentially compressed exocyclic F-C-F bond angle introducing additional strain energy. Consistent with this increased reactivity and following trends established to dale, the agents were found to be 500-1000x less cytotoxic than the corresponding CBI derivative lacking the difluorocyclopropane substitution. Similarly, the gem difluoro substitution had no perceptible effect on the DNA alkylation selectivity of the agents, and they were found to undergo the characteristic adenine N3 addition to the C9 cyclopropane carbon but did so with a reduced (675-725x) efficiency following the cytotoxicity and stability correlations.