The three-dimensional structures of a DNA oligonucleotide containing three extra unpaired adenosine residues (dGCCAGGAAATGGAC+dGTCCGACCTGGC) and that of the perfect duplex analogue (dGCCAGGTCGGAC+dGTCCGACCTGGC) have been studied in solution by 1H and 13C nuclear magnetic resonance. All non-exchangeable aromatic and H-1', H-2', H-2" sugar protons were assigned using standard assignment pathways for B-DNA. All cross-peaks within these pathways were present for the perfect duplex molecule as would be expected for a right-handed A or B-form duplex. However, a few cross-peaks which would be expected in the standard case are extremely weak in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of the bulged duplex even at long mixing times (250 ms). For example, almost no cross-relaxation is observed between the H-6 proton of C22 and the H-1' of A21, directly across from the three base bulge. Yet the continuity of assignment pathways through the three base bulge argues against any discontinuous "looping out" of one or more of the extra adenosine residues. Double quantum-filtered correlated spectroscopy experiments demonstrate very little deviation from south sugar conformations for residues at or near the bulge. The perfect duplex contains three A.T basepairs as expected, resulting in three very intense T imino-AH2 cross-peaks in the H2O NOESY experiment. In contrast, only two such intense cross-peaks are observed in the same experiment using the bulged duplex sample. Assignments of the two T imino peaks using one-dimensional NOEs are consistent with disruption of the T.A base-pair immediately 3' to the bulge; this is consistent with our earlier observation of chemical reactivity at a T 3' to an An or Tn bulge. We also find evidence of disruption of the G.C base-pair immediately 5' to the bulge.