We report three as yet undescribed, critical features of the interaction of the enediyne antitumor antibiotic esperamicin A1 (ESP A1) with DNA. First, results from hydrodynamic and spectroscopic studies revealed an intercalative binding mode conferred by the deoxyfucose-anthranilate of ESP A1. An intercalative binding mode is consistent with earlier observations of nucleosome linker-selective DNA damage by ESP A1 [Yu, L. et al. J. Biol. Chem. 1994, 269, 4144-4151]. While capable of adopting a planar structure, the N-(2-methoxyacrylyl) anthranilate group would represent an unusual intercalator, which by itself has no apparent affinity for DNA, yet contributes 1-2 kcal/mol to the binding energetics of ESP A1. Second, bistranded DNA lesions, which consist of a direct strand break opposite an abasic site, represent 20-25% of ESP A1-mediated DNA damage. Finally, sequencing gel analysis and tritium abstraction studies revealed that the deoxyfucose-anthranilate caused a switch in the chemistry of ESP A1-mediated DNA damage from 4’-hydrogen abstraction to 1’. We propose that intercalation of the anthranilate of ESP A1 alters the minor groove position of the diradical form of the drug and causes a switch in the hydrogen abstraction in bistranded lesions; the predominance of single-strand lesions associated with ESP A1 may be the result of the altered positioning or intramolecular quenching of one of the drug radicals.