We have purified the membrane-extrinsic anaerobic glycerol-3-phosphate dehydrogenase from Escherichia coli using a strain harboring a recombinant ColE1:E. coli plasmid carrying the glpA gene. The purified enzyme is composed of subunits of 62,000 and 43,000 molecular weight in 1:1 molar ratio as determined by medium dodecyl sulfate polyacrylamide gel electrophoresis, sedimentation equilibrium, and chemical cross-linking studies. The presence of 20% ethylene glycol stabilizes the enzyme by preventing dissociation of the subunits. The purified enzyme contains 1 mol of noncovalently bound FAD and 2 mol of non-heme iron/dimer. The FAD can be reduced by addition of substrate and resides in the large subunit. Addition of exogenous flavins stimulates the rate of the enzymatic reaction, and the effects of FAD and FMN can be distinguished by the following properties: (i) FAD causes a 20% increase in enzymatic activity with a half-maximal concentration of 200 nM whereas FMN results in a 6-fold increase in activity with a half-maximal concentration of 130 microM. (ii) When methylene blue replaces phenazine methosulfate as an oxidation-reduction coupling dye, FAD stimulates the rate of the reaction, whereas FMN inhibits it. Limited proteolysis or treatment with sulfhydryl reagents does not affect activity but removes capacity for stimulation by exogenous flavins.