The substrate specificity and stoichiometry of the reaction of a bacterial luciferase from Photobacterium phosphoreum were studied. This enzyme shows only negligible activity with FADH2 and reduced riboflavin, compared with that with FMNH2. Aliphatic aldehydes of longer chain length have higher affinity for the enzyme and caused a greater increase in the quantum yield of the bioluminescent reaction. The stoichiometry of the luciferase reaction was found to be luciferase: FMNH2: O2= 1: 1: 1.