The primary structure of the 2Fe-2S ferredoxin (protein B) from the benzene dioxygenase system of Pseudomonas putida strain NCIB 12190 was determined by gas-phase sequencing of the protein and its fragments. Fast atom bombardment mass spectrometry indicated a molecular mass of 11,860 Da. The sequence contained five cysteine residues, four of which would be required to coordinate the iron-sulphur cluster. The amino acid sequence determined in the present study is compared to that of a protein deduced from the DNA sequence from another strain of Pseudomonas putida. Little sequence homology was observed when protein B was compared to 2Fe-2S ferredoxins from plant and cyanobacterial sources. The novel sequence determined here suggests a new class of ferredoxin, which is consistent with the observed mid-point redox potential being significantly less negative (-155 mV) than those of the 2Fe-2S ferredoxins involved in photosynthesis (-310 to -455 mV).