Comparative study of the natural ligand effect on structural properties and conformational stability of human alpha-fetoprotein (AFP) and its homologue, human serum albumin (HSA), was performed using several approaches, including circular dichroism, fluorescence spectroscopy, and scanning microcalorimetry. Here we show that denaturation of AFP, induced by the increase of temperature or urea concentration, is irreversible. We have established the fact that this irreversibility is caused by ligand release from the AFP molecule. Interestingly, the ligand-free form of AFP has no rigid tertiary structure but exhibits substantial secondary structure and high compactness. This means that the rigid tertiary structure of AFP is controlled by interaction with ligands, while their release results in transition of a protein molecule into a molten globule-like intermediate. In contrast, processes of HSA denaturation and unfolding are completely reversible. Release of ligands from HSA results only in a small decrease in stability but not transformation into the molten globule state.