To explore the potentially available functional properties of β-lactoglobulin in, for example, the processing of food products, it is important to isolate the protein by a procedure that avoids all possible denaturing conditions, such as low pH, high ionic strength, or low or elevated temperatures that could cause the protein to undergo irreversible conformational changes. In this work, a mild isolation protocol for β-lactoglobulin from bovine milk is presented, applicable to semi large-scale isolations (50 to 200 g). The protein could be isolated with a high efficiency (>80€and a good purity (>98Ž Biochemical characterization of the material demonstrated no lactosylation of the protein, nor the formation of irreversibly associated dimers. Also, no proteose peptones could be detected. The ability of β-lactoglobulin to undergo conformational changes is studied by far and near-ultraviolet circular dichroism and differential scanning calorimetry. A βglobalβ unfolding of the protein is detected around 72 (tertiary level) and 77°C (secondary level). The dimer-monomer dissociation occurring around 52°C could also be monitored at a secondary structural level. Remarkably, a low temperature transition around 30°C was observed, where approximately 10 β-stranded residues unfold cooperatively, not been reported previously. This low temperature transition is irreversible at temperatures higher than 35°C or upon freezing the material at -20°C. The addition of 20␐lycerol could prevent this irreversible conformational change. The effect of the low temperature transition on the protein's functionality remains to be investigated.