1 Succinate dehydrogenase flavocoenzyme (“SD‐flavin”), previously shown to be an 8α‐substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 2 8α‐Histidyl‐riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH‐fluorescence curve and behavior on thin‐layer and paper chromatography, as well as paper electrophoresis. 3 Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)‐and N(3)‐imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6‐N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α‐N(3)‐histidyl‐riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin‐imidazole bond and identification of the methyl‐histidine liberated as 1‐methyl‐histidine.