Two 32 base pair, four-arm immobile Holliday junctions have been prepared and studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Two-quantum spectroscopy provides scalar (through bond) correlations for the 1'H, 2'H, and 2''H resonances of the deoxyribose sugar rings and the nonlabile cytosine and thymine base protons. Assignments in the deoxyribose sugars are extended to the 3'H resonances principally from relayed connectivities in total correlation spectra. Severe overlap of resonances in the standard regions of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitated the use of a unique approach for obtaining sequence-specific assignments of duplex DNA, wherein all possible NOE connectivities in the spectra are analyzed. These studies of 64-residue structures represent a substantial step forward with respect to the size of oligonucleotide for which virtually complete assignments are obtained. The assignments form the critical background for the detailed analysis of Holliday junction structure and dynamics that is required to address key issues in understanding the role of Holliday junctions in genetic recombination and repair.