Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in α-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the α-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap — 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing α-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of α-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of α-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of α-helix by proline. The average free energy change for the substitution of Ala by Pro in a α-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of ∼4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.