The three-dimensional structure of a disulfide-stabilized analog of the Dickerson/Drew dodecamer hairpin has been determined by NMR spectroscopy. The structural ensemble was determined from a set of conformations obtained from distance geometry calculations and optimized using restrained molecular dynamics and simulated annealing. The structures were further refined by iterative relaxation matrix approach calculations to include the effects of spin diffusion. To assess the quality of these structures, a signal-to-noise scaled "R" value has been defined that is based on the ratio of measured and back-calculated NOE cross-peaks. In the absence of motional dynamics, the group of ten structures with the lowest R values represents the group of structures that is most compatible with the NMR data. The stem region of the average structure forms a short helix, and the helical parameters and backbone angles fall within the range of observed values for B-DNA. The disulfide cross-link is not as defined as the stem or the loop because only a few experimental constraints are available for this region of the molecule; however, none of the conformations of the cross-link produced during the SA-IRMA refinement distorts the duplex geometry away from B-like DNA. The first three nucleotides in the loop stack over the 5’-end of the helix and are followed by a sharp turn at residue T8 which closes the loop. This pattern of loop folding is similar to that observed in studies of other DNA hairpins possessing tetranucleotide loops.