A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (∼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.