Subtilisin Carlsberg dissolved in aqueous solution is several orders of magnitude more active than the enzyme suspended in anhydrous acetonitrile. In order to ascertain why, we employed crystalline subtilisin lightly cross-linked with glutaraldehyde as a catalyst in both aqueous and organic media. The structure of this crystalline enzyme in acetonitrile had been previously found to be virtually identical to that in water, thus ruling out solvent-induced conformational changes as the cause of the enzymatic activity drop. Titration of the competent active centers of subtilisin revealed that k(cat)/K-M is solely responsible for this activity plunge. Quantitative mechanistic analysis of the 7-order-of-magnitude difference in k(cat)/K-M values between subtilisin dissolved in water and cross-linked subtilisin crystals suspended in anhydrous acetonitrile allowed accounting for at least 5.6 orders of magnitude. This drastic decline is due to (i) a marked shift in the activity vs pH profile of the cross-linked crystalline enzyme compared to its soluble counterpart; (ii) different (far less favorable in acetonitrile than in water) energetics of substrate desolvation; and (iii) very low thermodynamic activity of water in anhydrous acetonitrile resulting in a much more rigid and thus less active enzyme.