The electron paramagnetic resonance spectra of azoferredoxin and molybdoferredoxin, components of the nitrogenase of Clostridium pasteurianum, disappear when the proteins are oxidized by certain dyes. When molybdoferredoxin and azoferredoxin were mixed in a 1 to 2 molar ratio, the electron paramagnetic resonance spectrum of the mixture was the sum of the two spectra with the exception of a slight change in the azoferredoxin signal. Addition of magnesium ATP and dithionite to this reconstituted nitrogenase resulted in a rapid change in the spectrum of both nitrogenase components; the molybdoferredoxin spectrum at all g-values decreased with a half-life less than 70 ms to 40% of its original size whereas the azoferredoxin signal changed in shape and size with a half-life of less than 40 ms. If an ATP-generating system was added instead of MgATP so that no ADP accumulated, then the molybdoferredoxin signal almost completely disappeared and the azoferredoxin signal changed in shape and slightly in size. These changes occurred at molar ratios of molybdoferredoxin to azoferredoxin from 1:14 to 1:0.2. If the reaction was allowed to consume the reductant, then the molybdoferredoxin signal(s) was restored but the azoferredoxin signal disappeared. The signal of azoferredoxin was restored and the signal of molybdoferredoxin again disappeared on addition of more reductant. The data suggest that for nitrogenase to catalyze the reduction of substrates, the magnesium ATP-reduced azoferredoxin complex is formed first and this complex then reacts with molybdoferredoxin to allow electron flow. In addition the data suggests that the rate-limiting reaction is an ATP-mediated electron flow from azoferredoxin to molybdoferredoxin. Finally the results show that no flow of electrons from azoferredoxin or molybdoferredoxin occurs when a mixture of ADP and ATP in a molar ratio of 2:1 is added initially or is reached by conversion of ATP to ADP and inorganic phosphate during reduction of protons. A mechanism consistent with these findings is proposed.