Colyophilization with methyl-beta-cyclodextrin activates subtilisin Carlsberg by more than 200-fold in organic solvents, though this is a short-lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 degreesC) decreases exponentially with a t(1/2) of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the crosslinked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.