A second form of the NADPH-flavin reductase with an isoelectric point of 6.1 was purified to homogeneity from human erythrocytes. The enzyme showed NADPH-specific flavin reductase activity when FAD, FMN or riboflavin was used as an electron acceptor. Analyses of the amino acid compositions and immunological reactivities of the enzyme and the other flavin reductase with an isoelectric point of 8.1 revealed that the proteins of these two enzymes are indistinguishable to each other. Tightly bound NADP+, which was reducible by a NADPH-generating system, was specifically found in the second form of the enzyme.