The ferredoxin component (ferredoxinBPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography. The protein was a monomer with a molecular weight of 15,000 and contained 2 gram-atoms each of iron and acid-labile sulfur. Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm. The spectrum was partially bleached in the visible region upon reduction by reductaseBPH with NADPH as the source of electrons. Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein. Upon reduction with sodium dithionite, signals with gx = 1.82, gy = 1.92 and gz = 2.02 were detected. These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center. FerredoxinBPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductaseBPH to the terminal oxygenase during catalysis.