A time-resolved fluorescence anisotropy study on the molecular flexibility of active-site labeled anthraniloyl--chymotrypsin, dansylsubtilisin Carlsberg, and native subtilisin Carlsberg, suspended in organic solvents, is described. The internal rotational mobility of the fluorophore in the nanosecond time range could be separated from rotation of enzyme aggregates and rapid energy transfer processes. The enzymes suspended in dry organic solvents are less flexible than when dissolved in water. The enzyme flexibility increased with increasing hydration level. The results confirm that the increase in enzyme activity observed upon addition of low amounts of extra water is related to an increase in enzyme flexibility. Differences in enantioselectivity of subtilisin Carlsberg in different organic solvents have been correlated with differences in enzyme flexibility. The relationship between the internal rotational mobility of the fluorophore and the enantioselectivity provides the first experimental evidence that enzyme flexibility and enzyme enantioselectivity are correlated.