Small proteins generally fold cooperatively: disruption of significant parts of the folded structure leads to unfolding of the rest of the protein. We show here, using proline scanning mutagenesis, that the native-like tertiary fold of the alpha-lactalbumin (alpha-LA) molten globule is formed by the non-cooperative assembly of its constituent helices. In contrast to the drastic destabilizing effects of proline substitutions in cooperatively folded proteins, proline mutations in the molten globule appear to cause only individual helices to unfold, without significantly influencing the other helices or the overall topology. Thus, the key determinants of a protein's overall fold may not be of the all-or-none type.