The first step for evaluating the reliability of a docking algorithm is commonly known as "redocking" or "self-docking" simulation. This consists of extracting the coordinates of the co-crystallized conformation of a ligand, randomizing and redocking it to verify if the experimental binding mode can be reproduced . In the case of the P-gp/zosuquidar complex, this process is complicated by the ability of P-gp to bind two molecules of zosuquidar in the same pocket. Hence, we performed the redocking simulations in two different ways: first by removing both copies of the ligand and redocking them in the ligand-free binding site, and then by removing only one of the two copies and redocking it in the respective ligand-occupied binding site. Fig. shows the results of the redocking simulations. When using the ligand-free binding site, AutoDock Vina scoring function showed a preference to dock zosuquidar in position 2, with a root-mean-squared deviation (RMSD) of 5.3 Å, compared to the original conformation in the same position. While a high overall RMSD was obtained, the difluoro-tetracyclo-piperazine (DTP) moiety was well positioned by the docking algorithm (RMSD of this group alone: 0.8 Å), restoring the key interactions found in the bound conformation. On the contrary, docking the oxypropanol-quinoline moiety resulted to be more difficult, because of the large binding site size and the lack of constraints induced by the absence of the second copy of the inhibitor. Indeed, when using the two ligand-occupied binding sites, the accuracy of the redocking increased dramatically, with RMSD values of 1.1 Å and 1.8 Å, compared to the original positions 1 and 2, respectively.