By a combined gravimetric and ir technique, spectra of protein films are recorded during sorption isotherms at constant water content h (mg D2O/mg dry protein) in the range 0 les; h ⩽ 0.35 at 27 and 38°C. Computer-aided differential analysis shows the effect of progressive hydration on some significant sites of the protein such as the ionizable acidic side chains and the backbone amide carbonyls, as well as the spectrum of the adsorbed water itself. In order to derive thermodynamic properties of these sites, the measured sorption isotherm is decomposed in terms of a model which postulates the existence of two classes of primary sorption sites only, and these two contributions are independently checked by the ir data. The free energy of binding of the strong and weak binding sites is found to be 2.0 ± 0.2 and 0.40 ± 0.1 kcal/mol, respectively. A water-induced transition region is clearly detected in all the observed properties at 0.06 < h < 0.10 at 38°C and is shown to be due to changes involving both the structure of the absorbed water and the coverage of the absorption sites. A detailed picture of the hydration events is offered, and the relevance of these findings to protein dynamics is discussed.