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The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes., Grant BIO2005-04650 from the Spanish Ministry of Education and Science (MEC); the Commisariat à l’Energie Atomique; the French Centre National pour la Recherche Scientifique; the Université Joseph Fourier, Grenoble; the French Research Ministry through ANR NT05-4_42781 (to M.B.); ANR JCJC05-0077 (to B.B.); a return grant of the Junta de Andalucia (to A.I.A.); Lundbeckfonden and a long-term EMBO fellowship (to M.R.J.). J.L.O.R. and N.A.J.v.N. are recipients of an FPU and Ramón y Cajal research contract from the MEC, respectively; and the Access to Research Infrastructures activity in the 6th Framework Programme of the EC (Contract # RII3-026145, EU-NMR). Funding for open access charge: ANR NT05-4_42781.