NADH-cytochrome b5 reductase (b5R), present in various tissues of the body, is a redox enzyme of multiple functions. The deficiency of the enzyme leads to hereditary methemoglobinemia. With rabbit anti-b5R antibody for plate coating and enzyme-labeled anti-b5R monoclonal antibody as reporter, we have developed a sandwich ELISA procedure for the determination of b5R concentration. This procedure is sensitive to a wide range of linearity, and convenient in coping with large numbers of samples. Using this novel method, cytosolic b5R concentration in the erythrocytes of 30 normal Chinese adults was estimated to be 25.63±8.54 ng/mg Hb. It was found that the concentration of red cell soluble b5R of five newborns was significantly lower than that of normal adults and soluble b5R was undetectable in the erythrocytes of 4 patients with type I hereditary methemoglobinemia. Our results demonstrated that the reduced b5R activity in red cell cytosol of both neonates and type I hereditary methemoglobinemic patients results largely or mainly from the lowered b5R concentration. Our novel method might be further exploited for in-depth investigation of the relationship between the qualitative and quantitative changes of b5R with regard to physiological and pathological conditions.