For the first time, we applied a gas-phase electron diffraction (GED) method together with vibrational spectroscopy and quantum chemical calculations to investigate the equilibrium geometries of achiral meso and enantiomeric diastereomers of tetramezine [1,2-bis-(3,3-dimethyldiaziridin-1-yl)ethane] and their ratio in the mixture. In the joint structural analysis of these data, a new approach based on PES parameters is used in the framework of a static molecular model (small amplitude motion approximation). The agreement between the theoretical and experimental molecular intensities is characterized by a divergence factor Rf of 5.9%. The experimental re-parameters of tetramezine diastereomers agreed with our B3LYP/cc-pVTZ and MP2/cc-pVTZ calculations, which predicted the total energy of the meso form (Ci point group symmetry) to be lower than that of the enantiomeric form (C2 point group symmetry), by 6.4 and 4.7 kJ mol-1, respectively. The experimentally measured percentages of the meso and both enantiomeric diastereoisomers at 360 K were 70% and 30%, respectively. We characterized the meso form using 2D NMR spectra. Our GED data are in good agreement with the X-ray diffraction analysis of the meso form. This result reflects the weak effect of intermolecular interactions in the crystal. We assigned the IR spectrum bands of the crystalline meso form using the Pulay technique of scaling quantum chemical force fields. In the case of the enantiomeric form calculated at the same level, transferability of the refined scale factors was used for more reliable prediction of the mutual location and interpretation of its fundamental frequencies.