The in vitro biosynthesis of the cyanogenic glucoside taxiphyllin has recently been demonstrated in Triglochin maritima (Hösel, W., and Nahrstedt, A. (1980) Arch. Biochem. Biophys. 203, 753-757). We have now studied in more detail the multistep conversion of tyrosine into p-hydroxymandelonitrile, the immediate precursor of taxiphyllin, catalyzed by microsomes isolated from dark-grown seedlings. The biosynthetic pathway involves N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, and p-hydroxyphenylacetonitrile. In marked contrast to an analogous pathway in Sorghum bicolor, p-hydroxyphenylacetonitrile is the best substrate for cyanide production (Vmax = 224 nmol/h/g, fresh wt) and the physiological substrate tyrosine is the poorest (Vmax = 18.8 nmol/h/g, fresh wt). The substrates exhibit alkaline pH optima between 7.5 and 9, and all except tyrosine show pronounced substrate inhibition. We have found that p-hydroxyphenylacetonitrile generated in situ from tyrosine is free to equilibrate by diffusion with exogenous material. On the other hand, neither N-hydroxytyrosine nor p-hydroxyphenylacetaldoxime will readily exchange with exogenous intermediates. We consider both N-hydroxytyrosine and p-hydroxyphenylacetaldoxime to be channeled in T. maritima, whereas in S. bicolor N-hydroxytyrosine and p-hydroxyphenylacetonitrile are channeled and the aldoxime is freely exchangeable.