Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B 1–25 ). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B 1–25 was compared with that of a truncated homodimer (dSP-B 8–25 ) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S–S stretching region centered at ∼500 cm −1 indicated a stable, although highly strained disulfide conformation with a χ(CS–SC) dihedral angle of ±10° for the dSP-B 1–25 dimer. In contrast, the truncated dimer dSP-B 8–25 exhibited a series of disulfide conformations with the χ(CS–SC) dihedral angle taking on values of either ±30° or 85±20°. For conformations with χ(CS–SC) close to the ±90° value, the Raman spectra of the 8–25 truncated dimers exhibited χ(SS–CC) dihedral angles of 90/180° and 20–30°. In the presence of a lipid mixture, both constructs showed a ν(S–S) band at ∼488 cm −1 , corresponding to a χ(CS–SC) dihedral angle of ±10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼60–70° to the surface normal, while the β structure had ∼40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.