Firefly luciferase can catalyze the formation of fatty acyl-CoA via fatty acyl-adenylate from fatty acid in the presence of ATP, Mg 2 + and coenzyme A (CoA). A long chain fatty acyl-CoA (C 1 6 -C 2 0 ), produced by luciferase from a North American firefly (Photinus pyralis) and a Japanese firefly (Luciola cruciata), was isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Of a number of substrates tested, linolenic acid (C 1 8 : 3 ) and arachidonic acid (C 2 0 : 4 ) appear to be suitable for acyl-CoA synthesis. This evidence suggests that firefly luciferase within peroxisomes of the cells in the photogenic organ may be a bifunctional enzyme, catalyzing not only the bioluminescence reaction but also the fatty acyl-CoA synthetic reaction.