ΔG° Values for bovine alpha-lactalbumin (BAL) unfolding in a range of solvents, including simulated milk ultrafiltrate buffer, were determined. The denaturant used was guanidine hydrochloride. In Ca2+-free solvents fluorescence and ultraviolet (UV) difference absorbance unfolding profiles were non-superimposable leading to ΔG° estimates of 15–17 kJ mol−1 and 21–28 kJ mol−1, respectively. Therefore, the unfolding of BAL was consistent with a three-state reaction. The low and high ΔG° estimates may correspond to the loss of BAL 3° and 2° structure, respectively.The presence of excess Ca2+, two BAL unfolding transitions (I and II) were observed with both spectrophotometric techniques. Transition (II) was associated with a ΔG° value of 52 kJ mol− and is probably identical to a two-state BAL unfolding reaction described previously. Transition (I) appears to signify a novel feature of BAL structure.