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Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N -deacetylated AF lesion [ N -(2′-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N -acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the “ B -DNA” major groove, S is “stacked” into the helix with base-displacement, and W is “wedged” into the minor groove. Here, we conducted 19 F NMR, CD, T m , and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T m results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA–protein interaction.