The usefulness of the “NMR-window III” approach (see ref. 2e) has been demonstrated through the use of the deuterio isotopomeric 12mer oligo-DNA duplex obtained from protected partially deuterated 2′ ( R / S ), 3′, 5′ ( R / S )- 2H 3- 2′-deoxyribonucleoside blocks [see preceding paper). The 2D-NMR spectra of this deuterio isotopomeric DNA duplex I were compared with those of the natural counterpart (duplex II) and deuterium-labelled 12mer duplex III (prepared earlier by the “NMR-window II” approach, ref. 2e), and the results can be summarised as follows: (i) The simplification of the crosspeak pattern in H1′–H2′/2″ and H4′–H5′/5″ areas in the DQF-COSY spectra reduces the spectral crowding, thereby allowing a precise extraction of the coupling constants. (ii) The deuterio isotopomeric mixture of DNA duplex I provide hitherto unavailable nOe data sets which are sensitive to the conformational changes of the sugar moiety or the backbone. (iii) The extractable number of nOe constraints based on fully resolved crosspeaks and free from spin diffusion for deuterio isotopomeric dodecamer I are 308, whereas they are only 188 for the natural counterpart, suggesting the usefulness of the nOe crosspeaks in the semi-quantitative approach for the NMR-constrained structure refinement.