Samples of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) normally have two components, the fully active tyrosyl radical (Tyr(.)) and Fe-2(III)-containing protein (similar to 60%) and the Fe-2(III)-only met-R2 form (similar to 40%). Reaction with the 1- or multi- (maximum 4-) equiv reagent hydrazine under anaerobic conditions gives biphasic kinetics. From UV-vis absorbance changes, the first stage of reaction corresponds unexpectedly to reduction of the Fe-2(III) met-R2 component, rate constant 7.4 x 10(-3) M(-1) s(-1) (25 degrees C) at pH 7.5. The slower second stage is assigned as net reduction of Tyr(.) and one Fe-III of the Fe-2(III) center, rate constant 1.7 x 10(-3) M(-1) s(-1). Separate experiments with met-R2 protein, and previous evidence from EPR spectroscopy for the formation of an (FeFeIII)-Fe-II intermediate, support such a mechanism. Reduction of the second Fe-III is then rapid. The corresponding reduction of the Tyr122Phe R2 variant gives a rate constant of 7.7 x 10(-4) M(-1) s(-1), which is substantially (x 10) less than that for met-R2. This is in part explained by the decreased reduction potential of the variant. From pH variations in the range pH 6.6-8.5, N2H4 is the prime reactant with little or no contribution from N2H5+ (pK(a) 8.2). Phenylhydrazine (250 mV) is unable to reduce the Fe-2(III) center, and reacts only with the tyrosyl radical (a l-equiv process) of the active R2 protein (0.184 M(-1) s(-1)). The reaction is > 10(2) times faster than the 2-equiv N2H4 reduction of Tyr(.) and Fe-2(III). The pK(a) for C6H5N2H4+ is 5.27, C6H5N2H3 is the dominant species present under the pH conditions (6.5-8.5) investigated, and no pH dependence is observed. Contrary to a previous report, we conclude that the stability of the diimide (N2H2) does not allow separate studies of the reduction of the R2 protein with this reagent.