The enantioselectivities of subtilisin Carlsberg and Rhizomucor miehei lipase in organic solvents are found to strongly depend on the method by which the enzymes are prepared. For the transesterification between sec- phenethyl alcohol and vinyl butyrate in dioxane at 7"C, the enantioselectivity of subtilisin precipitated with isopropanol is more than twice that of the enzyme prepared by lyophilization from aqueous buffer. Furthermore, the temperature dependence of the enantioselectivity is influenced by the mode of enzyme preparation. For example, in the aforementioned process the enantioselectivities of subtilisin lyophilized from aqueous buffer ,and crosslinked subtilisin crystals increase when the temperature is raised from 7 to 45°C. In contrast, the enantioselectivities decrease with temperature forthe enzyme precipitated from aqueous solution with acetone or isopropanol and for the enzymatic hydrolysis in water. The temperature dependence of the enantioselectivity of subtilisin ly- ophilized from buffer is markedly affected by the solvent: In acetonitrile and nitromethane the enzyme is more enantioselective at higher temperatures, while negligible temperature effects have been found in tetrahydrofuran and pyridine. Lyophilized lipase exhibits striking temperature dependencies of its enantioselectivity in dioxane, acetonitrile, and nitromethane, while showing almost none in pyridine, triethylamine, and tetrahydrofuran. The results underscore the importance ofthe mode of enzyme recovery on enantioselectivity and its temperature dependence in enzymatic reactions in organic solvents (in contrast to those in water). 0