A novel ferredoxin was purified from Mycobacterium smegmatis by a series of hydrophobic chromatographies in the presence of high concentrations of ammonium sulfate and sodium chloride. The ferredoxin exhibited the same peptide map and N-terminal amino acid sequence as the known 7Fe ferredoxin from the same bacterium. On the other hand, this ferredoxin was found to contain approximately 6 Fe/mol ferredoxin and was also shown to contain only [3Fe-4S] clusters by resonance Raman spectroscopy, indicating that it is a novel 6Fe ferredoxin which contains two [3Fe-4S] clusters.