alpha-Hydroxynitrile lyase (HNL, acetone-cyanohydrin lyase, EC 4.1.2.37) was purified to homogeneity from petioles of cassava (Manihot esculenta Crantz). The purified HNL is a homotetramer with a subunit molecular weight of 25,600 and an isoelectric point of 4.7. The HNL activity exhibits a pH optimum of 5.0 and is stable in the pH range of 6 to 11. The petiole HNL shows a simple Michaelis-Menten kinetics with Km for acetone cyanohydrin of 4.0 +/- 0.9 mM and Vmax of 46.2 +/- 5.0 micromol/min/mg. Several alcohols, aldehydes, and ketones inhibit the HNL activity. The alcohols and ketones are competitive inhibitors, whereas the aldehydes are noncompetitive inhibitors. On the basis of Ki values, inhibitors with 4 carbons are more potent than those with the same functional groups but having fewer or more than 4 carbons.