BACKGROUND:In IR difference spectroscopy of proteins and biological systems, as in many other biophysical techniques, experiments at different hydration levels, and after H/D or H216O/H218O exchange are particularly important, for band assignment and for mechanistic studies. OBJECTIVE:Evaluate the kinetics, the effectiveness and the feasibility of an isopiestic approach for controlled hydration and isotopic exchange on bacterial photosynthetic reaction centers as a model for membrane proteins. METHODS:Mid-Infrared and Near Infrared spectroscopy experiments at different times and kinetic data treatment. RESULTS:Equilibria for hydration/dehydration, and for isotopic exchange are rapid (hour time scale) and can easily be applied in normal conditions. The exact hydration state and the effectiveness of isotopic exchange can be precisely assessed through FTIR or NIR spectroscopies. Furthermore, effectiveness of isotopic exchange (especially for internal exchangeable residues or molecules) appears to be higher compared to traditional methods. CONCLUSIONS:The isopiestic method can be easily applied to obtain controlled hydration conditions and high rate of isotopic exchange. This approach can be in principle applied to any biophysical technique.