N‐Acetyl‐2,3‐dehydroproline,N‐acetyl‐5‐oxo‐L‐proline,N‐acrylyl‐L‐proline,N‐acetyl‐L‐azetidine‐2‐carboxylic acid andN‐acetyl‐D,L‐pipecolic acid have been examined in2H2O by1H and13Cn.m.r. for the purpose of finding s‐cisor s‐translocked acylprolines. Conformationally locked acylprolines could be incorporated into proline‐containing peptide hormones such as angiotensin and thyroliberin in order to determine the rotational state of the peptide bond to proline in the hormone receptor complex. The populations oftransandcisrotational isomers were determined as a function of p2H in order to assign thetransandcisisomers and to compare the populations in all the acylprolines at neutral p2H, where thecisisomer is normally present. Proton spectra were also recorded at from 7° to 75° in order to qualitatively determine the exchange rate between the isomers. The majority of these analogs exhibit acis‐transisomerization similar to that ofN‐acetyl‐L‐proline in the ratio oftranstocisrotational isomer found at neutral p2H (about 1:1), the temperature dependence of the population ratio (none), and the coalescence temperature for proton resonances (greater than 75°). However,N‐acetyl‐5‐oxo‐L‐proline was found to be greater than 98% s‐transat neutral pH, compared to 50% s‐transinN‐acetyl‐L proline, and therefore a good candidate for synthesis of an s‐translocked peptide hormone.N‐Acetyl‐2,3‐dehydroproline rapidly exchanges between s‐cisand s‐transin contrast to all other proline analogs examined and exhibits coalescence of the β‐protoncisandtransresonances at 45°. Titration with the shift reagent Pr+++was employed to confirm the assignments of thecisandtransmethyl resonances of all of theN‐acetyl compounds exceptN‐acetyl‐5‐oxo‐L‐proline.