Human methionine synthase reductase (MSR) is a protein containing both FAD and FMN, and it reactivates methionine synthase that has lost activity due to oxidation of cob(I)alamin to cob(II)alamin. In this study, anaerobic redox titrations were employed to determine the midpoint reduction potentials for the flavin cofactors in two highly prevalent polymorphic variants of MSR, I22/L175 and M22/S175. The latter is a genetic determinant of plasma homocysteine levels and has been linked to premature coronary artery disease, Down's syndrome, and neural tube defects. The I22/L175 polymorphism has been described in a homocystinuric patient. Interestingly, this polymorphism is in the extended linker region between the two flavin domains, which may mediate or facilitate interaction with methionine synthase. In MSR I22/L175, the FMN potentials are - 103 mV (oxidized/semiquinone) and - 175 mV (semiquinone/hydroquinone) at pH 7.0 and 25°C, and the corresponding FAD potentials are -252 and -285 mV, respectively. For the M22/S175 variants, the values of the four midpoint potentials are -114 mV (FMN oxidized/semiquinone), -212 mV (FMN semiquinone/hydroquinone), -236 mV (FAD oxidized/semiquinone), and -264 mV (FAD semiquinone/hydroquinone). The midpoint potential values in the two variants are generally comparable to those originally determined for the MSR I22/S175 variant [Wolthers, K. R. (2003) Biochemistry 42, 3911-3920], with relatively minor variations in the different redox couples. In each case, blue neutral flavin semiquinone species are stabilized on both flavins, and are characterized by a broad absorption band in the long wavelength region. In addition, stopped-flow absorption and fluorescence spectroscopy were used to study the pre-steady state reduction kinetics by NADPH of the two polymorphic variants. The reversible kinetic model proposed for wild-type MSR was validated for the I22/L175 and M22/S175 variants. Thus, the biochemical penalties associated with these polymorphisms, which result in less effective methionine synthase activation, do not appear to result from differences in their reduction kinetics. It is likely that differences in their relative affinities for the redox partner, methionine synthase, underlie the differences in the relative efficiencies of reductive activation exhibited by the variants.