The membrane-associated pulmonary surfactant protein C (SP-C), containing a poly-valine -helix, and a synthetic SP-C analogue with a poly-leucine helix (SP-C(Leu)) have been studied by hydrogen/deuterium exchange (HDX) – matrix assisted laser desorption ionisation (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), forms abundant amyloid fibrils under experimental conditions. In CD3OD/D2O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchange 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N- and C-terminal heteroatoms and amide hydrogen atoms in the unstructured N-terminal regions. After approximately 300 h, 4 exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remain unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value, due to the formation of insoluble aggregates which are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period although the peptides were incubated together. In combination, HDX and aggregation data indicate that the poly-leucine peptide refolds into a helix after opening, while the unfolded poly-valine peptide forms insoluble -sheet aggregates rather than refolds into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which likely contributes to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo.