To study the changes in secondary, tertiary and quaternary structures and the alteration in protein–protein interactions during the protein aggregation upon heating, bovine serum albumin (BSA) was incubated over a range of temperature, and its structure was monitored by various biophysical techniques including circular dichroism (CD), second-derivative UV absorption spectroscopy, dynamic light scattering (DLS) and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS). The experimental data demonstrated that the size of BSA was relatively stable up to 60 1C. Above 65 1C, however, BSA underwent the obvious heat-induced aggregation (a dramatic growth of hydrodynamic radius Rh), accompanied with great unfolding of its secondary structure (a sigmoidal reduction of a-helix with the transition midpoint at 67.6 1C), with significant expansion of its tertiary structure (an increased solvent exposure of some aromatic residues with the transition midpoint at 65.5 1C), with distinct screening of electrostatic repulsion (a great reduction of second virial coefficient A2 above 65 1C) and with remarkable formation of aggregates (an evident increase of molecular weight above 65 1C).