The Holliday junction is a key intermediate in genetic recombination. It consists of four DNA strands that associate by base pairing to produce four double helices flanking a junction point. In the presence of multivalent cations, the four helices, in turn, stack in pairs to form two double-helical domains. The angle between these domains has been shown in a number of solution studies to be approximately 60 degrees in junctions flanked by asymmetric sequences. However, the recently determined crystal structure of a symmetric junction [Eichman, B. F., Vargason, J. M., Mooers, B. H. M., and Ho, P. S. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 3971-3976] finds an angle closer to 40 degrees, possibly because of sequence effects. From the crystal structure alone, one cannot exclude the possibility that this unusual angle is a consequence of crystal packing effects. We have formed two-dimensional (2D) periodic arrays of DNA parallelograms with the same junction-flanking sequence used to produce the crystals; these parallelograms are free to adopt their preferred interdomain angle. Atomic force microscopy can be used to establish the interdomain angle in this system. We find that the angle in this junction is 43 degrees, in good agreement with the results of crystallography. We have used hydroxyl radical autofootprinting to establish that the branch point is at the same migratory position seen in the crystals.