The ability of higher plants to catabolize phenolic substances has recently become well documented, but the early products of ring cleavage have mostly remained uncharacterized. Thus whereas the oxidation of 1,2 diphenols to muconic acids by intradiol ring cleavage is a familiar process in microorganisms, few instances have been reported where muconic acids are formed by plants or plant enzymes. One example of apparent occurrence of intradiol aromatic ring cleavage in flowering plants is the biosynthesis of the widely distributed cyanogenic glucoside triglochinin (1). Triglochinin (1) is expected to be formed by isomerisation of either of the two epimeric protriglochinins (2), which in turn may arise from 3 by intradiol ring fission. A preliminary biosynthetic study of 1 in the monocotyledon Triglochin maritima indicated its origin from tyrosine, the side chain of the amino acid being presumably modified by the general pathway known for other cyanogenic glycosides. Experiments with the dicotyledon Thalictrum aquilegiifolium, the major cyanogenic glucoside of which was formulated in effect as a monomethyl ester of 1, also broadly supported a pathway from tyrosine through 4 and 5 to 1. In this paper we report further results of feeding experiments with Triglochin maritima and Thalictrum aquilegiifolium. The plants were in early flower and contained 1 as the only evident cyanogenic constituent. After feeding with (RS)-tyrosine-[2-14C] and 4,5 or 6 labelled with 14C in the cyano group, 1 was isolated by CC on polyamide and ion-exchange chromatography, followed by repeated prep. PC. Since the label from the substrates was expected to be incorporated into the cyano group of 1, the glucoside was hydrolyzed enzymatically and the specific activity of the hydrogen cyanide evolved determined. The results demonstrate that all the precursors employed may be utilized in the biosyntheses of 1 in both plants. In general, the label was incorporated with high specificity except when the per cent incorporation was low. Of all substrates, 4 was most effectively converted into triglochinin. The cyanohydrins 5 and 6, which might decompose partially in the plant tissues with lower apparent incorporation as a consequence, were each utilized to a lesser degree. Our experiments demonstrate for the first time conversion of a 1,2-diphenol (6) to triglochinin, strongly supporting the involvement of 3.