A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. Similar to molecular chaperones, most of these compounds have no substrate specificity, but some specifically stabilize certain proteins. In the present work, the capacity of trehalose and glycerol, two well known osmolytes, to stabilize and renature inorganic pyrophosphatase is demonstrated. Both trehalose and glycerol significantly protect pyrophosphatase against thermoinactivation achieved by incubating the enzyme at temperatures up to 95 ^oC, displacing the optimal temperature and the denaturation curve to higher temperatures. Optimal temperature increased from 50^oC, in the absence of additives, to 60^oC, in the presence of 1 M trehalose or 25 % glycerol, to 70^oC, in the presence of 1.5 M trehalose or 37.5 % glycerol, and 80^oC in the presence of 2 M trehalose or 50 % glycerol. These effects occur in parallel to an increase in Q₁₀ of pyrophosphatase induced by increasing concentrations of both, trehalose and glycerol. Additionally, both osmolytes promote an increase in t_0.5 for inactivation of the enzyme incubated at 80 ^oC. Tertiary structure of pyrophosphatase, analyzed by intrinsic fluorescence measurements, revealed that trehalose and glycerol protect the unfolding of the enzyme induced by high temperature in the same extent that observed in catalytic activity measurements. Finally, the inactivation and unfolding observed after incubation of pyrophosphatase for long periods at high temperature is completely reversed when incubation is performed in the presence of trehalose and glycerol but not in the absence of osmolytes. To the best of our knowledge, there are no data on the effects of these compounds on renaturation of thermoinactivated proteins. The correlation between the recovery of enzyme activity and structural changes indicated by fluorescence spectroscopy contribute to better understanding of the protein stabilization mechanism.