NADH–cytochrome b 5 reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b 5 reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b 5 reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b 5 reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b 5 reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b 5 reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b 5 reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.