Double-stranded (ds)DNA formation and dissociation are of fundamental biological importance. The negative DNA charge influences the dsDNA stability. However, the base pairing and the stacking between neighboring bases are responsible for the sequence-dependent stability of dsDNA. The stability of a dsDNA molecule can be estimated from empirical nearest-neighbor models based on contributions assigned to base-pair steps along the DNA and additional parameters because of DNA termini. In efforts to separate contributions, it has been concluded that base stacking dominates dsDNA stability, whereas base pairing contributes negligibly. Using a different model for dsDNA formation, we reanalyze dsDNA stability contributions and conclude that base stacking contributes already at the level of separate ssDNAs but that pairing contributions drive the dsDNA formation. The theoretical model also predicts that stability contributions of base-pair steps that contain only guanine/cytosine, mixed steps, and steps with only adenine/thymine follow the order 6:5:4, respectively, as expected based on the formed hydrogen bonds. The model is fully consistent with the available stacking data and the nearest-neighbor dsDNA parameters. It allows assigning a narrowly distributed value for the effective free energy contribution per formed hydrogen bond during dsDNA formation of -0.72 kcal.mol(-1) based entirely on the experimental data.