The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction.