The second derivative FTIR study of heat-induced and pressure-assisted cold-induced changes in the secondary structure of bovine alpha-lactalbumin was carried out for native holoprotein and calcium ion depleted apoprotein. The secondary structure and compactness of alpha-lactalbumin were examined in a temperature range from 20 to 80 degrees C during the heat treatment and 20 to -15 degrees C during the pressure-assisted cold treatment. This was the first FTIR study on the pressure-assisted cold denaturation of a protein. Because protein solutions had close to neutral pD and low ionic strength, the apoprotein remained in the molten globule state and the holoform maintained its native tertiary structure. In order to distinguish between unfolding-related and partially deuterated exchange-related spectral changes, we examined both the fully deuterated holoform and the partially deuterated holoform. The quantitative analysis of the spectral changes in the amide I/I' vibrational band revealed that the 3(10) helices were more prone to thermal unfolding than the alpha helices. We observed that the protein's compactness and secondary structure were both considerably stabilized against an increase and decrease in temperature by the presence of a calcium ion. Under the conditions of this study, only the apoprotein was susceptible to the cold denaturation. In contrast to this, an unexpected linear increase of the alpha-helical content was observed upon the cooling of the holoprotein under high pressure. The results were discussed in reference to the existing crystallographic data for crystals of human alpha-lactalbumin grown at two different temperatures.