Using the photoreduced deazaflavin system, we examined the catalytic properties of biotin synthase of Bacillus sphaericus. In this assay system, only deazaflavin and fluorescent light replaced the complicated electron transport system essential for biotin synthase. The enzyme activity obtained from this assay system was five-fold higher than that obtained in the assay system using a cell-free extract of the wild-type strain, B. sphaericus IFO3525, as the electron transport system. Dithiothreitol (DTT) was essential for the enzyme reaction and the optimal concentration was 20 mM in the proposed assay system. Except for dithioerythritol, none of the other reductants tested, such as 2-mercaptoethanol, glutathione, and sodium dithionite could effectively replace DTT. Among metal ions, only ferrous ion was effective and the optimal concentration was 0.1 mM In this assay system. Compounds with two Or three phosphate groups, such as ATP, pyrophosphate and tripolyphosphate had significant inhibitory effects. This observation could give one possible strategy for the enhancement of microbial biotin production.