The calcium-depleted form of α-lactalbumin (α-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-α-LA at neutral pH. CD spectra revealed that the molten globule of apo-α-LA can be obtained upon mild heating at 45°C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-α-LA are essentially identical to those of the most studied molten globule of α-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-α-LA by proteinase K at 4°C occurs slowly as an all-or-none process leading to small peptides only. At 37°C, proteinase K preferentially cleaves apo-α-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the β-subdomain of the protein (chain region 34–57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-α-LA. A protein species given by the N-terminal fragment 1–34 linked via the four disulfide bridges to the C-terminal fragment 54–123 or 57–123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-α-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of α-LA maintains a native-like tertiary fold characterized by a rather well-structured α-domain and a disordered chain region encompassing the β-subdomain 34–57 of the protein. Proteins 2002;49:385–397. © 2002 Wiley-Liss, Inc.