Three-dimensional homology models of human MT1 and MT2 melatonin receptors were built with the aim to investigate the structure-activity relationships (SARs) of MT2 selective antagonists. A common interaction pattern was proposed for a series of structurally different MT2 selective antagonists, which were positioned within the binding site by docking and simulated annealing. The proposed antagonist binding mode to the MT2 receptor is characterized by the accommodation of the out-of-plane substituents in a hydrophobic pocket, which resulted as being fundamental for the explanation of the antagonist behavior and the MT2 receptor selectivity. Moreover, to assess the ability of the MT2 receptor model to reproduce the SARs of MT2 antagonists, three new derivatives of the MT2 selective antagonist N-[1-(4-chloro-benzyl)- 4-methoxy-1H-indol-2-ylmethyl]-propionamide (7) were synthesized and tested for their receptor affinity and intrinsic activity. These compounds were docked into the MT2 receptor model and were submitted to molecular dynamics studies, providing results in qualitative agreement with the experimental data. These results confirm the importance of the out-of-plane group in receptor binding and selectivity and provide a partial validation of the proposed G protein-coupled receptor model.