Stereo-selective deuteration has been explored as an approach for improving the accuracy of NMR-derived, three-bond vicinal proton-proton coupling constants in the 12-base-pair DNA Dickerson sequence [d(CGCGAATTCGCG) 2 ]. The coupling constants are useful for DNA structure determination in restrained molecular dynamics calculations. Specifically, the A5 and A6 residues were prepared with the H2؆ proton stereo-selectively replaced with a deuteron. Deuteration of the H2؆ leads to a 42-fold reduction in the transverse cross-relaxation rate of the H2 spin, effectively negating the contribution of transverse cross relaxation to the cross peak frequencies and phases. Calculated linewidth and polarization transfer functions indicated that the reduced dipolar interaction is also expected to result in a significant increase in intensity for all cross peaks involving the H1, H2, or H3 spin. The spectral complexity is also reduced by selective deuteration. Time-shared homonuclear decoupling of passive spins during acquisition was implemented, reducing the spin system, in some cases, to an effectively isolated two-spin system. This enables the use of a 90°mixing pulse instead of the 35°pulse commonly used in standard P.E.COSY experiments, leading to an additional 75% increase in signal intensity. Selective excitation pulses were used to reduce the number of increments required in the indirect dimension by as much as a factor of 4. The cumulative improvement in sensitivity is striking, approaching three orders of magnitude per unit time. Separate experiments, referred to as Stripe-COSY and Superstripe-COSY, were optimized for each coupling constant measured. Finally, J-doubling was used to obtain the most accurate peak separations. This comprehensive approach shows promise as an effective method for extracting highly accurate homonuclear vicinal coupling constants in DNA.