Enantiopure cryptophane-222 derivative (1) devoid of substituents was obtained via high-performance liquid chromatography (HPLC) using chiral stationary phases. The chiroptical properties of 1 were determined from polarimetry, electronic circular dichroism (ECD), synchrotron radiation circular dichroism (SRCD), vibrational circular dichroism (VCD), and Raman optical activity (ROA) experiments and were compared to those of the cryptophane-A (2) derivative. Unusual polarimetric results were obtained for 1 in CHCl3 solvent as the sign of the optical rotation (OR) values changes in the nonresonance region above 365 nm, whereas no change was observed in the CH2Cl2 solvent. ECD spectra in the L-1(a) and L-1(b) regions were very similar for the two solutions and could not explain these unusual polarimetric properties. In contrast, SRCD spectra in the B-1(b) region revealed spectral differences for the two solutions, which have been associated with conformational changes of the three linkers by time-dependent density functional theory (TDDFT) calculations. DFT calculations of the OR support that conformational changes may explain the polarimetric results obtained for the two solvents. Finally, TDDFT calculations of the ECD as well as DFT calculations of the VCD and ROA allowed the attribution of the (-)(589)-PP absolute configuration for 1 in solution, as determined from the X-ray structures of 1.