The biosynthetic pathway for the cyanogenic glucoside, dhurrin, in sorghum has previously been shown to involve the sequential production of (E) - and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the E-oxime to the Z-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the E-oxime into an S-alkyl-thiohydroximate with retainment of the configuration of the E-oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z-oximes but not E-oximes. The CYP79 derived E-oximes may play an important role in plant defense. This article is protected by copyright. All rights reserved.