A bioluminescent D-luciferin-luciferase mixture is separated by gel filtration during the time course of the reaction. A simultaneous analysis with an UV-visible diode array detector and an on-line luminometer gives nonsuperimposable chromatograms. Luminescence recordings display three peaks, one associated with the enzyme (light-emitting species 1: LES1), and two other species free from the luciferase: LES2, with a luciferyl-adenylate-like spectrum and LES3. Production of these two species is nucleotide (ATP or 2'-dATP)- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengths previously described (similar to 575 and similar to 620 nm).