The activity of an enzyme involved in decyanation of cyanocobalamin was found in the cell homogenate of Euglena gracilis. The enzyme essentially required FAD or FMN, and NADPH as cofactors. The apparent Km for cyanocobalamin and NADPH were 7.1 microM and 0.2 mM, respectively. The enzyme reaction obeyed allosteric kinetics towards FAD ([FAD]0.5 = 30 microM, n = 2.7; as calculated by the Hill plots). The Euglena enzyme was located in the mitochondria.