Using the bifunctional FAD synthetase from Corynebacterium ammoniagenes, which has the two sequential activities of flavokinase and FMN adenylyltransferase in FAD biosynthesis, a method of production of the intermediate FMN without any accumulation of FAD was investigated. Various phosphate polymers having no adenylyl moiety were tested for their ability to phosphorylate riboflavin to FMN, using a crude enzyme from C. ammoniagenes/pKH46, which is an FAD-synthetase-gene-dosed strain. Only metaphosphate, other than ATP, could phosphorylate riboflavin to FMN, but FAD did not accumulate at all, The conditions for the conversion of riboflavin to FMN were optimized. The metaphosphate-dependent phosphorylation reaction required Mg2+ as the most effective divalent cation. The best concentrations were 10 mM for MgCl2 and 3 mg/ml for metaphosphate. The riboflavin added to the reaction mixture was almost completely converted into FMN after 6 h incubation in the presence of high concentrations of the enzyme preparation.