Free-energy simulations have been used to estimate the change in the conformational stability of short polyalanine α-helices when one of the alanines is replaced by a proline residue. For substituting proline in the middle of the helix the change in free energy of folding (ΔΔG°) was calculated as 14 kJ/mol (3.4 kcal/mol), in excellent agreement with the one available experimental value. The helix containing proline was found to be strongly kinked; the free energy for reducing the angle of the kink from 40° to 15° was calculated, and found to be small. A tendency to alternate hydrogen bonding schemes was observed in the proline-containing helix. These observations for the oligopeptide agree well with the observation of a range of kink angles (18–35°) and variety of hydrogen bonding schemes, in the rare instances where proline occurs in helices in globular proteins. For substituting proline at the N-terminus of the helix the change in free energy of folding (ΔΔG°) was calculated as −4 kJ/mol in the first helical position (N1) and +6 kJ/mol in the second helical position (N2). The observed frequent occurrence of proline in position N1 in α-helices in proteins therefore has its origin in stability differences of secondary structure. The conclusion reached here that proline may be a better helix former in position N1 than (even) alanine, and thus be a helix initiator may be testable experimentally by measurements of fraction helical conformation of individual residues in oligopeptides of appropriate sequence. The relevance of these results in regards to the frequent occurrence of proline-containing helices in certain membrane proteins is discussed.