The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been studied in vitro by using [¹⁸O]oxygen and a microsomal enzyme system obtained from etiolated sorghum seedlings. The products formed were purified by HPLC and TLC, and the incorporation of [¹⁸O]oxygen was monitored by mass spectrometry. In the presence of NADPH and [¹⁸O]dioxygen, L-tyrosine is converted to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime with quantitative incorporation of an [¹⁸O]oxygen atom into the oxime function. The first step in this conversion is the N-hydroxylation of L-tyrosine to N-hydroxytyrosine. Administration of N-hydroxytyrosine as a substrate results in the production of 1-nitro-2-(p-hydroxyphenyl)ethane in addition to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, with quantitative incorporation of a single [¹⁸O]oxygen atom in all three products. These data demonstrate that the conversion of N-hydroxytyrosine to p-hydroxyphenylacetaldehyde oxime involves additional N-hydroxylation and N-oxidation reactions giving rise to the formation of 2-nitro-3-(p-hydroxyphenyl)propionate, which by decarboxylation produces aci-1-nitro-2-(p-hydroxyphenyl)ethane. Both compounds are additional intermediates in the pathway. The two [¹⁸O]oxygen atoms introduced by the N-hydroxylations are enzymatically distinguishable as demonstrated by the specific loss of the oxygen atom introduced by the first N-hydroxylation reaction in the subsequent conversion of aci-1-nitro-2-(p-hydroxyphenyl)ethane to (E)-p-hydroxyphenylacetaldehyde oxime. A high flux of intermediates through the microsomal enzyme system is obtained with N-hydroxytyrosine as a substate. This renders the conversion of the aci-nitro compound limiting and results in its release from the active site of the enzyme system and accumulation of the tautomeric nitro compound.