Conformational transitions of holo-alpha-lactalbumin in a hydro-ethanolic cosolvent system was studied by spectrofluorescence, CD in near- and far-uv regions, and high-sensitivity differential scanning calorimetry. Experimental results allow us to propose that in isothermal conditions alpha-lactalbumin undergoes a number of conformational transitions with increasing ethanol concentration: N<=>I<=>D<=>H. The existence of I-state was deduced from spectrofluorometric and near-uv CD data. In this state the aromatic chromophores of the amino acid side chains are more accessible to the solvent displaying higher local mobility. The H-state was detected from far-uv CD spectra as a state corresponding to the content of alpha-helices higher than originally found in native protein. However, calorimetric measurements provide data revealing only the two-state mechanism of alpha-lactalbumin unfolding in both water and in aqueous ethanol solutions. This indicates that the energy levels of N- and I-states as well as of D- and H-states are similar. Thermodynamics of the unfolding of alpha-lactalbumin in hydroethanolic solutions was analyzed with the help of the linear model of solvent denaturation. Unfolding increments of enthalpy, entropy, and Gibbs energy of transfer of the protein from a reference aqueous solution to hydro-ethanolic solutions of different concentrations were determined from the calorimetric data. They are linear functions of molar ethanol fraction. The slope of the unfolding increment of Gibbs energy of transfer was calculated from data on transfer of amino acid residues taking into account the average solvent accessibility of amino acid residues in the native structure of small globular proteins, using the additive group contribution method.