To establish the substrate specificity of the thioglucoside glucohydrolase myrosinase (EC 22.214.171.124), this enzyme was purified to homogeneity from light-grown cress (Lepidium sativum L.) seedlings by Sephadex gel filtration. Red Dye and anion exchange (FPLC Mono Q) chromatography, and preparative isoelectric focusing. Hydrolytic activity was shown toward only 4 of the 29 synthetic and natural O-and S-glycosides tested. Highest activity was displayed with the endogenous glucosinolates benzylglucosinolate (K m , 295 μM) and sinigrin (K m , 300 μM) at an optimum pH of 5.5 in sodium citrate buffer. The synthetic glycosides PNPG (K m , 2.0 mM) and ONPG were poorer substrates at an optimum pH of 6.5 in potassium phosphate buffer. The enzyme was inactive with all other nitrophenyl glycosides tested including PNP-a-D-glucoside and PNP-thio-β-D-glucoside, suggesting a requirement for O-β-D-glucose as the glycone moiety within these substrates. PNPG hydrolysis was stimulated 2.6-fold by ascorbate (1 mM). The enzyme exhibited no metal ion requirement and was strongly inhibited by lead nitrate, mercury chloride, and ferric chloride at 1 mM concentration. The metal chelators DIECA , EDTA , o-phenanthroline, and 2,2′-dipyridyl were not inhibitory, but the thiol reagents PCMS, PCMB, and N-ethylmaleimide (at 1mM) caused 50 -80% inhibition of enzyme activity.