Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular H-1 paramagnetic relaxation enhancement (PRE) arising from Mn2+ chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn2+) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular H-1-PRE profiles are observed on the H-1 resonances of the bound protein when dT-EDTA-Mn2+ is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular 1H-PRE data demonstrate unambiguously that HMGB-1 A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the 1H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a sermiquantitative estimate of the equilibrium populations at the various sites.