Polyubiquitin (polyUb) chains function as signaling molecules that mediate a diverse set of cellular events. The outcome of polyubiquitination depends on the specific linkage between Ub moieties in the chain, and differently linked chains function as distinct intracellular signals. Although an increasing number of Ub-binding proteins that transmit the regulatory information conferred by (poly)ubiquitination have been identified, the molecular mechanisms of linkage-specific signaling and recognition still remain to be understood. Knowledge of the chain structure is expected to provide insights into the basis of diversity in polyUb signaling. Here we describe several NMR approaches aimed at determining the physiological conformation of polyUb and characterization of the chains' interactions with ubiquitin-binding proteins.