The binding of aflatoxin B1 to DNA and DNA adduction by aflatoxin B1 exo-8,9-epoxide were studied as a function of DNA conformation. Equilibrium binding of aflatoxin B1 to A-, B-, and Z-form helices was monitored by measurement of NMR linewidth for the methoxy protons of aflatoxin B1. The data revealed that as compared to B-form DNA, the association of aflatoxin with the A-form helix is significantly reduced. No binding to Z-DNA was observed. The yields obtained following reaction of the respective monomers with aflatoxin B1 exo-8,9-epoxide revealed that only dG forms an adduct. The reactivity of aflatoxin B1 exo-8,9-epoxide with single-strand DNA was determined via constant-temperature experiments in which the yield of adduct was measured for a family of oligonucleotides of increasing thermal stability. The results demonstrate that duplex structure favors adduct formation. Adduct yields were compared for A-, B-, and Z-form helices. About 12 times less adduct is produced from the A-form helix as compared to B-form, while no adduct is produced from a Z-form duplex. The results support the conclusion that reaction of aflatoxin B1 exo-8,9-epoxide with DNA proceeds via an intercalated transition-state complex only with the B-form double helix.