Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of β-lactoglobulin under various physicochemical conditions. The effects of pH (3–9), NaCl (0–2 M), and lactose, glucose and sucrose (100–500 g/l) in the temperature range 25–100 °C on the conformation sensitive amide I band in the i.r. spectrum of β-lactoglobulin in D2O solution were examined. The 1692 cm−1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of β-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm−1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm–1, assigned to β-sheets. The 1692 cm−1 band was therefore attributed to a β-type structure. β-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 °C), β-lactoglobulin was partly denatured. Heating to 60–80 °C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm−1, characteristic of intermolecular β-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of β-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of β-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.