2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF-induced SMIs formed during translesion synthesis. We performed spectroscopic, thermodynamic, and molecular dynamics studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and −1, −2, and −3 deletion duplexes of the 5′-CTCTCGATG[FAAF]CCATCAC-3′ sequence and an additional −1 deletion duplex of the 5′-CTCTCGGCG[FAAF]CCATCAC-3′ NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being ‘GC’–1 (73%) > ‘AT’–1 (72%) > full (60%) > −2 (55%) > −3 (37%). Thermodynamic stability was in the order of −1 deletion > −2 deletion > fully paired > −3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs is thermodynamically more stable than the conformationally flexible external B conformer. Results from the molecular dynamics simulations indicate that perturbation of base stacking dominates the relative stability along with contributions from bending, duplex dynamics, and solvation effects that are important in specific cases. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.