In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich α-helix comprising residues 9–34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The α-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10°C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains β-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol–1, in which the polypeptide segment 9–27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 °C. The α-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.