The study investigates the contributions of base stacking and base pairing to the thermal stability of the DNA double helix. By examining DNA molecules with solitary nicks and gaps, the authors measure the temperature and salt dependence of the stacking free energy of the DNA double helix. They find that base-stacking interactions are the primary stabilizing factor in the DNA double helix, while A•T pairing is always destabilizing and G•C pairing contributes minimally to stabilization. The temperature and salt dependences of the stacking term fully determine the temperature and salt dependence of DNA stability parameters. The study also reveals that the heterogeneity of stacking interactions in A•T- and G•C-containing contacts significantly contributes to the sequence-dependent stability of DNA polymers. These findings provide a paradigm shift in the understanding of the interplay of forces stabilizing the DNA double helix.The study investigates the contributions of base stacking and base pairing to the thermal stability of the DNA double helix. By examining DNA molecules with solitary nicks and gaps, the authors measure the temperature and salt dependence of the stacking free energy of the DNA double helix. They find that base-stacking interactions are the primary stabilizing factor in the DNA double helix, while A•T pairing is always destabilizing and G•C pairing contributes minimally to stabilization. The temperature and salt dependences of the stacking term fully determine the temperature and salt dependence of DNA stability parameters. The study also reveals that the heterogeneity of stacking interactions in A•T- and G•C-containing contacts significantly contributes to the sequence-dependent stability of DNA polymers. These findings provide a paradigm shift in the understanding of the interplay of forces stabilizing the DNA double helix.