1-Methyladenine (m1A) alters T.A Watson-Crick to T.m1A Hoogsteen base pair. Owing to its conversion to N6-methyladenine (m6A) at higher temperatures, thermodynamic studies of m1A-containing DNAs using conventional melting methods are subject to the influence of m6A species. In this study, we applied nuclear magnetic resonance spectroscopy to determine the base pairing modes and effect of m1A on thermodynamic stability of double-helical DNA. The observed base pairing modes account for the destabilizing trend which follows the order T.m1A approximately G.m1A<A.m1A<C.m1A, providing insights into the m1A flipping process and enhancing our understandings of the mutagenicity of m1A.