The paper introduces HADDOCK, a protein-protein docking approach that utilizes biochemical and/or biophysical interaction data, such as chemical shift perturbation data from NMR titration experiments or mutagenesis data, to drive the docking process. These data are introduced as Ambiguous Interaction Restraints (AIRs), which define ambiguous intermolecular distances between residues involved in the interaction. The accuracy of HADDOCK is demonstrated using three molecular complexes: the N-terminus domain of Enzyme I (EIN) with histidine-containing phosphocarrier protein (HPt), Enzyme IIA β-glucose (E2A) with HPt, and HIV protein gp120 with CD4. For two of these complexes, both the complex and free protein structures were solved, and NMR titration data were available. For the gp120-CD4 complex, only X-ray structures of individual partners were used, and mutagenesis data were employed. In all cases, the best structures generated by HADDOCK, defined by the lowest intermolecular energies, were closest to the published structures in terms of backbone root-mean-square deviations (iRMSD). The results show that HADDOCK can effectively use ambiguous interaction restraints to guide the docking process, improving the accuracy of the predicted structures.The paper introduces HADDOCK, a protein-protein docking approach that utilizes biochemical and/or biophysical interaction data, such as chemical shift perturbation data from NMR titration experiments or mutagenesis data, to drive the docking process. These data are introduced as Ambiguous Interaction Restraints (AIRs), which define ambiguous intermolecular distances between residues involved in the interaction. The accuracy of HADDOCK is demonstrated using three molecular complexes: the N-terminus domain of Enzyme I (EIN) with histidine-containing phosphocarrier protein (HPt), Enzyme IIA β-glucose (E2A) with HPt, and HIV protein gp120 with CD4. For two of these complexes, both the complex and free protein structures were solved, and NMR titration data were available. For the gp120-CD4 complex, only X-ray structures of individual partners were used, and mutagenesis data were employed. In all cases, the best structures generated by HADDOCK, defined by the lowest intermolecular energies, were closest to the published structures in terms of backbone root-mean-square deviations (iRMSD). The results show that HADDOCK can effectively use ambiguous interaction restraints to guide the docking process, improving the accuracy of the predicted structures.