The in vivo synthesis of firefly luciferin is not completely understood. Only the final step of the enzymatic pathway has been studied, which is the condensation reaction of -cysteine with 2-cyano-6-hydroxybenzothiazole, and is the same reaction used to produce the compound synthetically. This was confirmed by radiolabeling of atoms in the two compounds and by identification of a luciferin-regenerating enzyme.
In firefly, oxidation of luciferins, which is catalyzed by luciferases, yields a peroxy compound 1,2-dioxetane. The dioxetane is unstable and decays spontaneously to carbon dioxide and excited ketones, which release excess energy by emitting light (bioluminescence).
Firefly luciferin and modified substrates are fatty acid mimics and have been used to localize fatty acid amide hydrolase (FAAH) in vivo. Firefly luciferin is a substrate of the ABCG2 transporter and has been used as part of a bioluminescence imaging high throughput assay to screen for inhibitors of the transporter.