The conformational space of 1,4,7-trioxacyclononane (9-crown-3) was studied using Conflex-AMBER and ab initio calculations at the density functional level of theory (DFT). The Conflex-AMBER calculations predict that the [225](100) conformation with C-2 symmetry is the global minimum with the [144](0) conformation being 0.9 kcal mol(-1) above the global minimum and these two conformations exist in a deep conformational well. The next lowest energy structure is 3.3 kcal mol(-1) above the global minimum, The DFT studies show that the stability order is inverted. The C-2 symmetry conformation [225](100) is 0.9 kcal mol(-1) higher in energy than the global minimum [144](0) conformation and the next lowest energy conformer is 4.8 kcal mol(-1) above the global minimum. The study highlights the flexibility of 9-crown-3 and the suitability of Conflex-AMBER program for conformational space searching of macrocyclic Ligands.