C 2 H 5 NO 2 ) Stereochemistry (rotational isomerism) Conformers/rotamer (trans, gauche, cis) Steric effects (torsion angle) Basis set limit (BSL) Benchmark (DFT, DFT-D, LC-DFT) a b s t r a c t A complete understanding of the conformational equilibria of proteogenic amino acids (AAs) is the first step towards understanding the 3D structures of peptides and proteins. High-level ab initio electronic structure calculations, including extrapolations to the complete basis set (CBS) limit, resulted in a highly precise energy description of the conformational equilibrium in gas-phase alanine. Twelve structures with different torsion angles were analyzed. The CCSD(T)/CBS relative energies of alanine (Ala, CH 3 CH(NH 2 )COOH) conformers were estimated using CCSD(T)/aug-cc-pVTZ, MP3/aug-cc-pVQZ, and MP2/aug-cc-pV5Z calculations at DFT-B3LYP/aug-cc-pVTZ geometries. Core correlation shifts are also included. An accuracy of 0.03-0.30 kcal mol Γ1 (10-105 cm Γ1 ) has been reached. The first three conformations of alanine were found to be almost isoenergetic. The possible reasons and consequences of this finding are discussed. A comparison of these data with glycine data is provided. A correlation between the C@O bond length of alanine conformers and their relative energies was found. Two groups of conformers (low-and high-energy) were identified. The importance of an accurate and systematic quantum-chemical description of intramolecular hydrogen bonding in gaseous (free) alanine is discussed. Benchmark data for other (simpler) modern quantum chemistry methods is provided (e.g., density functional theory with dispersion correction or DFT-D). The data presented can deepen our current understanding of the sophisticated potential energy surface (PES) of protein building blocks -amino acids -and the factors stabilizing their rotational isomers.