NADH:cytochrome b5 oxidoreductase catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. Utilizing a heterologous expression system for the soluble, catalytic domain of the rat microsomal enzyme, we have produced two mutants, corresponding to E255- and G291D. These mutants correspond to the two specific mutations that were identified over a half century later following diagnosis of the original cases of type I recessive congenital methemoglobinemia (RCM). We have purified both the E255- and G291D variants to homogeneity to determine the molecular basis for type I RCM in these individuals. Both the E255- and G291D variants retained a full complement of FAD and exhibited absorption and CD spectroscopic properties comparable to those of the wild-type protein. Oxidation-reduction potentiometric titrations yielded standard midpoint potentials (E0') for the FAD/FADH2 couple of -271 and -273 mV for the E255- and G291D variants, respectively, which were comparable to the value of -268 mV obtained for the wild-type protein and confirmed that the redox potential of the flavin was unaffected by either mutation. Thermal and proteolytic stability studies revealed that while the G291D variant exhibited stability comparable to that of wild-type, the E255- variant was markedly less stable, indicative of an altered conformation. Initial-rate kinetic studies revealed that both mutants had decreased catalytic activity (kcat), with the E255- and G291D variants retaining approximately 38 and 58% of wild-type activity, respectively. However, the affinity for NADH (KmNADH) was decreased approximately 100-fold for E255- compared to only approximately 1.3-fold for G291D, results supported by the spectroscopic binding constant (Ks) obtained for G291D. These results indicate that the properties of both the E255- and G291D cytochrome b5 oxidoreductase mutants are similar to those of other variants that have been identified as resulting in the type I form of RCM.