Riboflavin binding (or carrier) protein (RfBP) is a monomeric, two-domain protein, originally purified from hens' egg white. RfBP contains nine disulfide bridges; as a result, the protein forms a compact structure and undergoes reversible three-state thermal denaturation. This was demonstrated using a differential scanning calorimetry (DSC) method [Wasylewski M. (2000) J. Prot. Chem. 19(6), 523-528]. It has been shown that the RfBP complex with riboflavin denaturates in a three-state process which may be attributed to sequential unfolding of the RfBP domains. In case of apo RfBP, the ligand binding domain denaturates at a lower temperature than the C-terminal domain. Ligand binding greatly enhances the thermostability of the N-terminal domain, whereas the C-terminal domain thermostability is only slightly affected and, in case of the examined holo RfBPs, the denaturation peaks of both domains merge or cross over. The magnitude of the changes depends on ligand structure. A detailed study of protein concentration effects carried out in this work allowed to estimate not only the thermostability of both domains but also the strength of domain interactions. The DC p , of denaturation was found for C-terminus and N-terminus of RfBPriboflavin complex to amount to 2.5 and Γ1.9 kcal mol Γ1 , respectively. The calculated domain interaction free energy, DG CN , was estimated to be approximately Γ1580 cal mol Γ1 at 67.0 8C. This value indicates that the interdomain interaction is of medium strength.