A microsomal system catalyzing the in vitro synthesis of the aglycones of the two cyanogenic glucosides linamarin and lotaustralin has been isolated from young etiolated seedlings of cassava (Manihot esculenta Crantz). A prerequisite to obtain active preparations is the complete removal of the endosperm pellicle covering the cotyledons before seedling homogenization. The rates of conversion of the parent amino acids valine and isoleucine to their cyanohydrins are 19 and 6 nmol/h/mg protein, respectively. The conversion rates for the corresponding oximes (2-methylpropanal oxime and 2-methylbutanal oxime) are 475 and 440 nmol/h/mg protein and for the nitriles (2-methylpropionitrile and 2-methylbutyronitrile) 45 and 75 nmol/h/mg protein. With the exception of 2-cyclopentenylglycine, none of the additionally tested amino acids are metabolized, whereas a broad substrate specificity is observed using oximes and nitriles as substrates. The in vitro biosynthesis is photoreversibly inhibited by carbon monoxide, demonstrating the involvement of cytochrome P450 in the hydroxylation processes. All tissues of the cassava seedling contain cyanogenic glucosides. The microsomal enzyme system responsible for their synthesis is restricted to the cotyledons and their petioles. This demonstrates that the cyanogenic glucosides are actively transported to other parts of the seedling. The enzyme activity decreases with the height of the etiolated seedling and is barely detectable in seedlings above 75 mm.