Attention is focused on L-5,5-dimethylproline (dmP) as a substitute to lock L-proline (Pro) in a cis conformation in peptides and proteins, to prevent cis/trans isomerization when a protein with cis X-Pro peptide groups unfolds. Procedures have been developed to obtain optically pure L-dmP and to incorporate this sterically hindered residue as tbe central one in tripeptides that are suitable for fragment coupling to prepare synthetic proteins. Based on the sequences of residues 92-94 (Tyr-Pro-Asn:YPN) and 113-115 (Asn-Pro-Tyr: NPY) in bovine pancreatic ribonuclease A (RNase A), in which the X-Pro peptide groups are in the cis conformation, the tripeptides Ac-Tyr-dmP-Asn (YdmPN) and Ac-Asn-dmP-Tyr (NdmPY) were synthesized, and their structures were determined by 2D H-1 nuclear magnetic resonance (NMR) spectroscopy. YdmPN was found to exist solely in the cis conformation between 6 and 60 degrees C, whereas NdmPY was found to have some trans component that increased from about 10% to about 21% as the temperature increased over the range between 6 and 80 degrees C. Both YdmPN and cis-NdmPY adopt a type VI reverse turn, as does proline. The NMR structures of YdmPN and cis-NdmPY are comparable with the X-ray structures of the corresponding portions of RNase A, and the NMR structure of trans-NdmPY is compatible with the X-ray structure of the isolated tripeptide, Ac-NPY. These results demonstrate that L-dmP is a promising substitute for proline in a variety of protein problems to constrain the X-Pro peptide group to the cis conformation.