Hydrogen-deuterium exchange for streptavidin and its complex with biotin is studied by means of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. To analyze the spectral changes upon deuteration, difference spectra and two-dimensional correlation spectra are calculated. We find that the exchange rate varies with the secondary structure in which the exchanging amide protons are incorporated. The most slowly exchanging protons, with a characteristic time constant on the order of hours, are part of the β-sheet secondary structure. The separation in time of exchange of the β-sheet from other structural elements allows the amide II and II' frequencies of the β-sheet (1530 and 1445 cm-1) to be identified. A second component which exchanges more rapidly than the β- sheet is characterized by its amide I frequencies 1680, 1640, and 1465 cm- 1. This component is attributed to the exchange of amide groups in secondary structures other than the central β-barrel. Binding of the ligand results in a slower exchange rate of the rapid component. These changes are interpreted in terms of structural differences observed by previous X-ray studies of one loop in the protein involved in the binding of the ligand. The presence of the ligand is found to inhibit the exchange of ~10 amide protons during the time of the experiment (10 h). The protected amide groups are most likely part of the β-sheet structure, and their retarded exchange is tentatively interpreted in terms of a reduced flexibility of the tetrameric protein upon binding of the ligand.