Comparing the behavior of freely suspended and immobilized enzyme in organic media with low water contents can yield insights into interactions among the biocatalyst, solvent, and support that influence protein structure and function. Immobilized chymotrypsin has higher activity than the suspended enzyme (by 1-2 orders of magnitude) in anhydrous organic solvents ranging from nonpolar n-octane to polar acetonitrile. In anhydrous tetrahydrofuran (THF), glass-adsorbed chymotrypsin is ca. 10 times more active than the suspended enzyme, and election spin resonance (ESR) spectra of an active-site spin-label reveal greater local flexibility. Upon adding up to 0.5% v/v water, increased catalytic efficiency of the immobilized enzyme is accompanied by a sharp rise in active-site polarity but no apparent change in active-site conformation or dynamics. Under the same conditions the activity of the suspended enzyme also increases; however, the active-site polarity remains nearly constant while the spin-label reflects increasing molecular flexibility. For both preparations, further changes in protein structure occur as enzyme activity increases with 0.5-7% v/v added water. Computer simulations of the room-temperature ESR spectra suggest that different initial conformational states contribute to the different behavior of the two enzyme systems over the entire range of added water. These findings show that the structural properties of suspended and immobilized enzyme can differ markedly and that these differences are important to enzyme activity in organic media.