Improved methods have been devised for the isolation in μmole quantities of a pure flavin pentapeptide and its acid‐hydrolysis product (SD‐flavin) from inner‐membrane preparations of heart mitochondria and from soluble, purified succinate dehydrogenase. SD‐flavin differs from riboflavin in still having an amino acid covalently linked to the isoalloxazine ring system. SD‐flavin may be compared with riboflavin and with various 8α‐substituted synthetic flavins by optical spectrophotometry in the neutral and cationic states and by ESR and ENDOR spectrometry in the cationic radical state. On the basis of these experiments is was concluded that the FAD prosthetic group of mitochondrial succinate dehydrogenase is covalently linked through the 8α‐position to the peptide backbone of the protein. This conclusion is in accord with the acid stability of the natural product and its tendency to yield riboflavin under reductive conditions. The unusual pH‐fluorescence spectrum of the flavin strongly suggests that the 8α‐methylene group is linked to an amino acid through a tertiary nitrogen group.