The synthesis and biological evaluation of penicillamine 6 -5-tert-butylproline 7 -oxytocin analogs and comparison with their proline 7 -oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen 1 ,Pen 6 ]-oxytocin (1, pA 2 = 8.22, EC 50 = 6.0 nM) and [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-oxytocin (2, pA 2 = 8.19, EC 50 = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using 1 H NMR, laser Raman and CD techniques. In H 2 O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln 4 and Pen 6 in the major trans-conformer of 1 as well as for Gln 4 in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 ± 0.2 kcal/mol) for isomerization of the amide N -terminal to 5-tBuPro 7 relative to OT was calculated from measuring the coalescence temperature of the Gly 9 backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C β -S-S-C β dihedral angles of +110-115°and ±90°, respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a χ CS−SC dihedral angle value close to 115°was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a ±90°angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a ±90°χ CS−SC dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114°and +116°. Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104°. In sum, the conformation about the disulfide in [dPen 1 ,Pen 6 ]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro 7 (5R)-tert-butyl group, the Leu 8 isopropyl sidechain and the Pen 6 β-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110°may be a prerequisite for their binding at the receptor.