The conformational propensities of the proline analogue bearing a phenyl substituent attached to the β carbon, in either a cis or a trans configuration relative to the carbonyl group, have been investigated. The behaviour of cis- and trans(βPh)Pro has been compared with that of proline in homochiral and heterochiral dipeptide sequences. NMR and IR studies as well as X-ray diffraction analysis provide evidence that the β-phenyl substituent does not disrupt the tendency of proline to occupy the i+1 position of a β-turn. The puckering of the pyrrolidine ring is significantly affected by the presence of the aromatic substituent, which tends to occupy positions that minimize steric repulsions. As a consequence, this substituent adopts specific well-defined orientations, which are more restricted for the cis derivative. Interactions between this aromatic group and that in the adjacent phenylalanine residue may be responsible for some of the conformational differences observed among the different peptides studied.