A microsomal fraction from Trifolium repens L. shoots of genotype Ac Ac has been used in the study of the biosynthesis of the two glucosides, linamarin and lotaustralin. It was demonstrated that it is likely that one set of enzymes is responsible for the biosynthesis of both hydroxynitriles from their respective amino acid precursors. The nitrile precursor of linamarin, 2-methylpropane nitrile appears to be used much less effectively than the oxime precursor. The results are discussed in relation to the role of the Ac/ac locus in the genetic control of cyanogenic glucoside biosynthesis, and in relation to results obtained with Linum usitatissimum.