Trifluoroethanol (TFE) is known to stabilize the alpha-helical structure in proteins and their fragments. However, the relationship between the TFE-induced structures and the native structure is not clear. Here we show that beta-lactoglobulin, which consists predominantly of beta-sheets, exhibited a markedly high propensity to form an alpha-helical structure in the presence of TFE, as measured by far-UV circular dichroism. A cooperative transformation from the beta-sheet structure to an alpha-helical structure occurred at a TFE concentration between 10% and 20%. These results were in contrast to a gradual beta-sheet to alpha-helix transition of the constant fragment of the immunoglobulin light chain, which is also a beta-sheet protein. To understand the significance of the high helical propensity of beta-lactoglobulin, we measured the TFE-induced conformational transition of more than 20 proteins of various secondary structural types. Whereas the alpha-helical proteins showed a propensity to form an extensive helical structure in TFE, the helical propensity of proteins with a low helical content in the native state varied. The helical content in TFE was correlated more with the helical content predicted by a secondary structure prediction than with the helical content of the native structure, suggesting that the stability of the helical structure in TFE is determined by local interactions between nearby amino acid residues. Our results suggest that an alpha-helical intermediate can accumulate during the refolding process of beta-lactoglobulin and that a hierarchical model of protein folding is not necessarily true for some beta-sheet proteins including beta-lactoglobulin.