Vibrational absorption and vibrational circular dichroism spectra of avidin with and without d-biotin in phosphate buffer were recorded in the amide I ' (1800-1600 cm(-1)) region as a function of temperature. The differences in the unfolding pathway of avidin in the presence and absence of biotin were examined using the curve-fitting results of absorption spectra, and the variable-temperature absorption and VCD spectra. This study reveals, contrary to previous spectroscopic studies, but in agreement with X-ray structural studies, that avidin contains beta -sheet structures with turns and bends, but does not contain a-helical structure. Also a cooperative structural transition leading to formation of aggregated antiparallel beta -strands, with increasing temperature, has been inferred. In avidin-biotin complex, however, some reversible unfolding of beta -sheet structure is noted but a cooperative structural transition has not been noted with increase in temperature. Two-dimensional (2D)-VCD correlation spectroscopy has also been used to analyze the sequence of events in structural unfolding of avidin.