(S)-Hydroxynitrile lyase (EC 4.1.2.39) from Hevea brasiliensis (rubber tree) catalyzes the reversible cleavage of cyanohydrins to aldehydes or ketones and prussic acid (HCN). Enzyme kinetics in both directions was studied on a model system with mandelonitrile, benzaldehyde, and HCN using two different methods-initial rate measurements and progress curve analysis. To discriminate between possible mechanisms with the initial rate method, product inhibition was studied. Benzaldehyde acts as a linear competitive inhibitor against mandelonitrile whereas HCN shows S-linear I-parabolic mixed-type inhibition. These results indicate an Ordered Uni Bi mechanism with the formation of a dead-end complex of enzyme, (S)-mandelonitrile and HCN. Prussic acid is the first product released from the enzyme followed by benzaldehyde. For progress curve analysis, a kinetic model of an Ordered Uni Bi mechanism including a dead-end complex, enzyme inactivation, and the chemical parallel reaction was set up, which described the experimental values very well. From the reaction rates obtained the kinetic constants were calculated and compared with the ones obtained from the initial rate method. Good agreement could be achieved between the two methods supporting the suggested mechanism.