In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D-QSAR studies. This hypothesis was used in a ligand-based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 microM, and among them two compounds exhibited sub-micromolar K(i) values. These results validated our applied methodologies, and the two compounds with sub-micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.