Three [5-t-BuPro(7)]oxytocin analogues were synthesized by substituting (2S,5R)-5-tert-butylproline for proline in oxytocin, [Mpa(1)]oxytocin, and [dPen(1)]oxytocin. Relative to oxytocin, [5-t-BuPro(7)]oxytocin and [Mpa(1),5-t-BuPro(7)]oxytocin exhibited strongly reduced binding affinity to the receptor; however, both peptides maintained the pharmacophore characteristics responsible for signal transfer evoking the same maximal response as oxytocin in the single-dose procedure and exhibiting partial agonistic activity in the cumulative dose-response procedure. Although [dPen(1)]oxytocin exhibited inhibitory as well as partial agonistic activity, [dPen(1),5-t-BuPro(7)]oxytocin exhibited only inhibitory potency with a similar in vitro pA(2) value of 7.50 in the absence of magnesium. In the presence of magnesium, [dPen(1), 5-t-BuPro(7)]oxytocin exhibited stronger inhibitory potency than [dPen(1)]oxytocin and no partial agonism. Assignment of the proton signals for the 5-tert-butylprolyl amide cis- and trans-isomers by two-dimensional NMR experiments in water indicated that the Cys(6)-Pro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured respectively at 35%, 33%, and 20% in the 5-tert-butylproline(7) analogues of oxytocin, [Mpa(1)]oxytocin and [dPen(1)]oxytocin. Although caution must be taken when relating the increase in cis-isomer population with an influence on biological activity in [5-t-BuPro(7)]oxytocin analogues, the synthesis and evaluation of analogues 1-3 have provided additional evidence that can be used to support the hypothesis that the prolyl amide cis-isomer may favor antagonism and the trans-isomer is necessary for agonist activity.