SummaryFNR, the activator of anaerobic respiratory genes ofEscherichia coli, has previously only been isolated as a protein ofMr, 29 000, which lacks nineN‐terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation.The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr, 30 000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr, 29 000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. InE. colistrain CAG627, proteolysis was greatly reduced making it possible to isolate FNR ofMr, 30 000. TheN‐terminal sequence of FNR (Mr, 30 000) was identical to that predicted from thefnrgene starting with the initiating methionine residue and including a four‐cysteine cluster (16)Cys–X3–Cys–X2–C