The process of DNA double-strand (dsDNA) formation for a four-base pair (dCGCG)(2) model system was studied using umbrella sampling combined with replica-exchange molecular dynamics simulations (REMD) and a generalized Born continuum solvent model. Disruption of dsDNA during the simulations was achieved by stepwise increasing the reference distance in a quadratic restraining potential between the nucleic acid backbone of the two DNA strands. During the reverse simulation (stepwise decrease of the distance starting from completely separated and unfolded single strands) full reformation of a dsDNA in close agreement with B-form geometry was achieved during REMD but not continuous MD simulations. The simulations allowed the calculation of a potential of mean force for the dsDNA formation along the reaction coordinate and were used to characterize intermediate structures. In addition, it was possible to analyze the change of various energetic contributions during disruption and formation of dsDNA that favor or disfavor duplex formation. The calculated free energy change of approximately -3.2 (+/-1.5) kcal mol(-1) and enthalpy change of approximately -37 kcal mol(-1) for (dCGCG)(2) duplex formation was in good agreement with corresponding experimental values of approximately -3.9 kcal mol(-1) and -38.5 kcal mol(-1), respectively.