A 32-base-pair model of the Holliday junction (HJ) intermediate in genetic recombination has been prepared and analyzed in-depth by 2D and 3D (1)H NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permutation of the base pairs at the junction relative to a previously studied HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11459], designed to probe the effect of the sequence at the n - 1 position (where n is the residue directly at the branch point) on the stacking geometry. Observation of several interbase nuclear Overhauser effects (NOEs) clearly indicates a strong preference for the isomer opposite that observed for J2, confirming the dependence of stacking isomer preference on the sequence at the junction. As for other model HJs studied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single (15)N-labeled thymine residue at the branch point. 1D (15)n-filtered (1)H-detected experiments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more direct and accurate measure of the crossover isomer distribution. The comparative analysis of our immobile HJs and a model cruciform structure [Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the relevance of crossover isomer preference in vivo.