L-Methionine-35S and DL-cysteine-35S were good sources of sulfur for the biosynthesis of sinigrin in Armoracia lapathifolia Gilib. and were incorporated with approximately the same efficiency. Homomethionine-35S was a slightly poorer source of sulfur than methionine, while taurine-35S was a very poor source. When homomethionine, methionine, and cysteine were employed as sources of sulfur, the distribution of radioactivity between the two sulfur atoms in sinigrin was approximately 80% in the isothiocyanate moiety and 10–15% in the sulfate moiety. When taurine was employed, the distribution was reversed. 1-Thio-β-D-glucose (1-thioglucose) also was a poor source of sulfur for sinigrin. The carbon skeleton of 1-thioglucose was only slightly incorporated into this thioglucoside. The experiments indicated that 1-thioglucose was not a direct precursor of the 1-thioglucosyl residue of sinigrin.The administration of doubly labeled methionine-2-14C-35S clearly demonstrated that this amino acid was not incorporated intact into sinigrin. The carbon-2 and sulfur atoms were metabolized by two different routes.Preliminary studies related to sulfur incorporation into the thioglucosides of Nasturtium officinale R.Br., Tropaeolum majus L., and Reseda luteola L. indicated that there were some differences depending on the source of sulfur; DL-cysteine was a better source of sulfur than either methionine or 1-thioglucose. Tropaeolum majus L. appeared to utilize the sulfur of cysteine for the production of thioglucoside as efficiently as Armoracia lapathifolia Gilib.; however, the other two species utilized the sulfur from the amino acids very poorly.