Comparison of the amino acid sequences of several microsomal cytochrome P-450 reductases to the flavoprotein domain (BMR) of cytochrome P-450BM-3 has revealed that this class of flavoproteins contains evolutionarily conserved regions that are important for their interaction with nucleotide substrates and cofactors. In order to understand the properties of BMR, the region encoding this protein, beginning at residue Lys-472 of cytochrome P-450BM-3, was subcloned and expressed in Escherichia coli. The recombinant protein (more than 50% of host-soluble proteins) was purified to homogeneity using conventional purification procedures. BMR (Mr 66,000) showed typical flavoenzyme absorbance spectra, contained FAD and FMN in a stoichiometry of 1:1, and catalyzed reduction of several artificial electron acceptors with rates comparable to those of the microsomal NADPH-cytochrome P-450 oxidoreductase. Limited trypsinolysis of BMR, under non-denaturing conditions, revealed that the protease removed the NH2-terminal 122 residues. This region was postulated to contain amino acids that are important for FMN binding (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158). Consistent with this hypothesis, the major tryptic product of BMR (BMR-52, Mr 52,000) contained only FAD, in an equimolar ratio to the protein. Also, like the FMN-depleted microsomal NADPH-cytochrome P-450 oxidoreductase (Kurzban, G. P., Howarth, J., Palmer, G., and Strobel, H. W. (1990) J. Biol. Chem. 265, 12272-12279), BMR-52 was active for only catalyzing ferricyanide reduction. These data provide strong experimental evidence for a discrete multidomain structure of BMR, as proposed for the membrane-bound reductases, with an amino-terminal FMN binding region and carboxyl-terminal FAD- and NADPH binding regions. Thus, BMR strongly resembles the microsomal cytochrome P-450 reductase and offers an opportunity to better understand the structure-function relationships of this class of flavoproteins.