Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K(m), 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 +/- 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid-Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-beta-d-glucoside (K(m), 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-beta-d-glucoside (O-glucosidase activity) with K(i) values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.