This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-(αMe)Ser(Ψ^H,HPro)-Ala-OBu^t (6a) and Fmoc-Ala-(S)-(αMe)Ser(Ψ^H,HPro)-Ala-OBu^t (6b). Additionally, the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is additionally influenced by the presence of two symmetrically independent molecules in an asymmetric unit. Both molecules adopt a conformation which resembles β-turn type II, stabilized by hydrogen bonding. The conformational preferences and prolyl cis–trans isomerization of Ac-(αMe)Ser(Ψ^H,HPro)-NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the amide bonds, with lower puckering in the transition state of the cis–trans isomerization.