We have determined the three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), a DNA duplex found in the promoter region of a gene regulating sporulation in a Bacillus subtilis mother cell, utilizing two-dimensional nuclear Overhauser effect (2D NOE) spectra. Once we assigned the cross-peaks in NOESY spectra, we calculated interproton distances from the NOE intensities by using the program MARDIGRAS. We then used this distances to calculate the structure of our duplexes by independently using two different methods: restrained molecular dynamics and Monte Carlo simulations. No matter which method we used, we obtained essentially the same structure lending us confidence that our final structure is defined by experimental restraints and does not depend significantly on the refinement method used. This structure shows some of the features that have been described for DNA duplexes in solution, namely some helical parameters exhibit some characteristics of A-DNA rather than being strictly in the B-DNA family of structure as one might expect for DNA in solution. While solving the structure of this duplex, we realized that some of the NOE intensities are affected by fast conformational dynamics intrinsic of the DNA duplex. These inconsistencies are nonlinear and can be used to draw insights into the dynamic nature of DNA duplexes structure. By using methods such as molecular dynamics with weighted time averaged restraints (MD-tar) and free Monte Carlo simulations to sample conformational space, we concluded that the inconsistencies observed in NOE derived base to sugar intraresidue distances in DNA can be reconciled with a two-state jump anti/syn sugar repuckering process combined with a restricted diffusion (rocking) about the glycosidic bond. The use of the Computer Graphics Laboratory resources, and in particular the molecular graphics program MidasPlus, has been an essential part of the methodology used to accomplish our goals.