The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin-bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin-binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in the mitochondrial Fp subunit suggests their functional importance.