Mycena chlorophos is a species of molecular oxygen-dependent bioluminescent fungus, and its pileus gills emit bright green light. The chemical mechanisms underlying this bioluminescence phenomenon are not yet understood. An enzyme (luciferase) producing light from trans-3-hydroxyhispidin is present in M. chlorophos pileus gills. However, it is unclear whether trans-3-hydroxyhispidin is an actual bioluminescence substrate (luciferin) in the natural bioluminescence of M. chlorophos. In the present study, this question is resolved. It was clearly demonstrated that the trans-3-hydroxyhispidin analog trans-3-hydroxybisnoryangonin significantly inhibited the artificial luminescence induced by the addition of trans-3-hydroxyhispidin to living pileus gills but did not inhibit natural bioluminescence in living pileus gills. This inhibition was due to the reaction of trans-3-hydroxybisnoryangonin with luciferase for trans-3-hydroxyhispidin. Even though trans-4-aminocinnamic acid is known to inhibit natural bioluminescence in living pileus gills, in the present study, trans-4-aminocinnamic acid did not influence the artificial luminescence via trans-3-hydroxyhispidin in the presence of luciferase for trans-3-hydroxyhispidin. These inconsistencies between the natural bioluminescence and the artificial luminescence of trans-3-hydroxyhispidin indicate that trans-3-hydroxyhispidin is not an actual luciferin in natural bioluminescence. Trans-3,4-dihydroxycinnamic acid is generally known to be an intermediate in trans-3-hydroxyhispidin biosynthesis. The artificial luminescence induced by the addition of trans-3,4-dihydroxycinnamic acid to living pileus gills was not inhibited by trans-3-hydroxybisnoryangonin. Therefore, trans-3,4-dihydroxycinnamic acid does not contribute to the luminescence involving trans-3-hydroxyhispidin in living pileus gills. (C) 2018 Elsevier Inc. All rights reserved.