Time-resolved fluorescence resonance energy transfer has been used to examine the global structure and conformational flexibility of three-way DNA junctions containing unpaired bases at the branch point. Three-way junctions were prepared with donor (fluorescein) and acceptor (tetramethyl-rhodamine) dyes attached to the ends of different helical arms in various pairwise combinations. The time-resolved fluorescence decay of the donor in each labeled junction was measured by time-correlated single photon counting. The distributions of donor-acceptor (D-A) distances present between each pair of labeled helices were recovered from analysis of the donor decay profiles using a Gaussian distribution model. The recovered D-A distance distributions reveal the mean distance between each pair of helices, as well as the range of distances that exists between each pair. For the junction lacking unpaired bases, the three mean interarm distances are similar, indicating an extended structure. In addition, a relatively broad range of distances is present between each pair of helices, showing that the structure is flexible. The addition of unpaired bases causes the junction to fold into a different structure, with one interarm distance being shorter than the other two. The change in overall geometry of the junction appears to be primarily due to the repositioning of one of the helices flanking the bulge. In bulged junctions containing unpaired thymine, cytosine, or adenine bases, the helix containing the 3' portion of the bulged strand appears to undergo the greatest change in its mean position relative to the other helices. In contrast, in the bulged junction containing unpaired guanine bases, the helix containing the 5' portion of the bulged strand is displaced. In all bulged junctions, there is a wide range of distances between the perturbed helix and the other two helices, indicating high mobility for the perturbed arm. These results indicate that the overall structure and conformational flexibility of three-way DNA junctions are sensitive to the presence of unpaired bases at the branch point of the junction and that the precise effect of a bulge depends on the nature of the unpaired bases.