Enzyme structure and function have been studied for subtilisin BPN’ solubilized in organic solvents by ion pairing with low concentrations of an anionic surfactant (Aerosol OT) in the absence of reversed micelles. Soluble subtilisin shows strikingly different behavior in octane and tetrahydrofuran (THF). In octane, the k(cat)/K-m for the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) is 370 M(-1) s(-1), within one order of magnitude of the enzyme’s hydrolytic activity in water. Moreover, the observed half-life of the soluble enzyme in octane is nearly three orders of magnitude greater than in water, presumably because of the absence of autolysis in the organic solvent. In contrast, the catalytic efficiency of the enzyme dissolved in the polar solvent THF is 0.04 M(-1) s(-1), and the enzyme loses 99% of its activity within 10 min. Comparable enzyme inactivation could also be observed in octane, but only at elevated temperatures such as 70 degrees C. Therefore, the mechanisms of deactivation of the soluble enzyme were investigated in both octane and THF. Kinetic and spectroscopic (CD and EPR) studies support the existence of multiple inactive forms of the soluble enzyme in THF at 25 degrees C and in octane at 70 degrees C. Notably, in both cases a denatured form can be renatured in anhydrous octane at 25 degrees C, the first demonstration of enzyme renaturation in a bulk organic solvent. A model explaining the THF- and thermally-induced inactivation processes of soluble subtilisin BPN’ is proposed, and the apparent reasons for the exceptionally high activity and stability of the soluble enzyme in octane are discussed.