The nonlabile protons of two 32-base-pair models of the Holliday junction intermediate in genetic recombination have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. The sequence of these models is designed to fully inhibit branch migration of the junction and to probe the possible sequence dependence of these four-arm DNA structures. Overlap of resonances in homonuclear two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multipathway approach for obtaining sequence-specific assignments, wherein all possible NOE connectivities are analyzed in parallel. Using this strategy, 1H resonance assignments were obtained for virtually all nonlabile base protons and C1', C2', and C3' sugar protons. Several unambiguous cross-arm NOE connectivities were identified, directly establishing the stacking arrangements of each contiguous (two-arm) helical domain. The distribution of the two possible stacking isomers is distinctly different for the two junctions studied, thereby indicating that the relative stability of the isomers is dependent on the sequence at the junction.