(S)-hydroxynitrile lyase from Hevea brasiliensis (EC 4.1.2.39) catalyzes the reversible formation of cyanohydrins from aldehydes of ketones and HCN. Stability and activity of hydroxynitrile lyase were investigated in citrate-phosphate buffer as well as in 11 different two-phase systems of buffer and water-immiscible organic solvents with a lag P ranging from 0.6 to 3.5. The formation of (S)-mandelonitrile from benzaldehyde and HCN was studied as a model system. Hydroxynitrile lyase is inactivated by its substrates and inhibited by the product; therefore, high partition coefficients for substrates and products are necessary to keep the concentration in the aqueous phase low. Enzyme stability and activity in the two-phase systems strongly depend on the solvent but do not correlate with the log P. The kinetics of the model reaction was monitored in different two-phase systems following the conversion and the enantiomeric excess of (S)-mandelonitrile. Both parameters are strongly dependent on the solvent used and are affected by enzyme stability From the organic solvents studied, the highest conversion as well as the highest enantiomeric excess could be obtained in a two-phase system of diisopropyl ether and buffer.