A three-disulfide form of human alpha-lactalbumin, with free thiols on Cys6 and Cys120, can adopt the molten globule conformation. It then spontaneously rearranges its three disulfide bonds to many isomers that tend to maintain the molten globule conformation. The distribution of free thiol groups within the rearranged species has been determined quantitatively by chemical modification and peptide mapping. The protein's eight cysteine residues were modified with nearly equal frequencies, although there were significant departures from randomness. The results confirm that the molten globule state of alpha-lactalbumin does not maintain the native-like topology of the polypeptide backbone but is more like a collapsed form of an unfolded protein.