The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b-558 (the membrane anchor protein) has recently been established. We have now determined the extent of N-terminal processing of each polypeptide by radiosequence analysis. At the same time, direct evidence for the correctness of the predicted reading frames has been obtained. The cytochrome showed a ragged N-terminus, with forms lacking one residue, and is inserted across the membrane without an N-terminal leader-peptide. Covalently bound flavin was not detectable in B. subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N-terminal processing of the apoprotein. This provides an explanation to why the succinate dehydrogenase synthesized in E. coli is not functional and demonstrates that host-specific factors regulate the coenzyme attachment.