The question of whether lipases can be activated by adsorption onto an interface in organic solvents was addressed using Rhizomucor miehei lipase as a model. In aqueous solution, this enzyme was shown t o undergo a marked interfacial activation. However, lipase (either Iyophilized or precipitated from water with acetone) suspended in ethanol or 2-(2-ethoxyethoxy)ethanol containing triolein exhibited no jump in catalytic activity when the concentration of triolein exceeded its solubility in these solvents, thereby resulting in formation of an interface. To test whether the lack of interfacial activation was due to the insolubility of the enzyme in organic media, lipase was covalently modified with poly(ethy1ene glycol). The modified lipase, although soluble in nonaqueous media, was still unable t o undergo interfacial activation, regardless of the hydrophobicity of the interface. This inability was found t o becaused by the absence of adsorption of lipase onto interfaces in organic solvents, presumably because of the absence of the hydrophobic effect (the driving force of lipase adsorption onto hydrophobic interfaces in water) in such media. The uncovered lack of interfacial adsorption and activation suggests that the short a-helical "lid" covering the active center of the lipase remains predominantly closed in nonaqueous media, thus contributing t o diminished enzymatic activity.