Conformational distributions of a four-way DNA junction have been examined by time-resolved fluorescence resonance energy transfer (FRET). A series of dye-labeled junctions were synthesized with donor (fluorescein) and acceptor (tetramethylrhodamine) dyes conjugated to the 5' termini of the duplex arms in all six pairwise combinations. The fluorescence decay of the donor in each junction was measured by time-correlated single-photon counting. The distributions of donor-acceptor (D-A) distances present between each pair of arms were recovered from the donor decays using a continuous Gaussian distribution model. The overall geometry of the four-way junction defined by the six mean D-A distances was consistent with a stacked-X structure, wherein pairs of duplex arms associate to form two continuous domains. Large differences were observed in the widths of the D-A distance distributions, depending on which pair of arms were labeled with the donor and acceptor dyes. Distances measured along the stacking domains were characterized by relatively narrow distributions, indicating that these domains were rigid, whereas distances between stacking domains had broader distributions, reflecting variability in the angle between the two domains. The distances described by broad distributions were overestimated by steady-state FRET measurements. These results suggest that an ensemble of stacked-X structures are present in solution, characterized by differences in the small angle between the stacking domains. Temperature and solvent effects on the recovered distribution widths provide an indication of flexibility in the four-way junction.