AbstractThe molecular structures of the two lowest‐energy conformers of proline, Pro‐I and Pro‐II, have been characterized by ab initio electronic structure computations. An extensive MP2/6‐31G* quartic force field for Pro‐I, containing 62 835 unique elements in the internal coordinate space, was computed to account for anharmonic vibrational effects, including total zero‐point contributions to isotopomeric rotational constants. New re and improved r0 least‐squares structural refinements were performed to determine the heavy‐atom framework of Pro‐I, based on experimentally measured (A. Lesarri, S. Mata, E. J. Cocinero, S. Blanco, J. C. Lopez, J. L. Alonso, Angew. Chem. 2002, 114, 4867; Angew. Chem. Int. Ed. 2002, 41, 4673) rotational constant sets of nine isotopomers and our ab initio data for structural constraints and zero‐point vibrational (ZPV) shifts. Without the ab initio constraints, even the extensive set of empirical rotational constants cannot satisfactorily fix the molecular structure of the most stable conformer of proline, a 17‐atom molecule with no symmetry. After imposing the ab initio constraints, excellent agreement between theory and experiment is found for the heavy‐atom geometric framework, the root‐mean‐square (rms) residual of the empirical rotational constant fit being cut in half by adding ZPV corrections. The most significant disparity, about 0.07 Å, between the empirical and the best ab initio structures, concerns the r(N⋅⋅⋅H) distance of the intramolecular hydrogen bond. Some of the experimental quartic centrifugal distortion constants assigned to Pro‐II have been corrected based on data obtained from a theoretical force field.