The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 μM oxyluciferin; 0.0025 to 1.25 μM L-AMP) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 μM ATP and D-Luciferin (from 3.75 up to 120 μM). Nonlinear regression analysis with the appropriate kinetic models (Henri–Michaelis–Menten and William–Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (K_i = 0.50 ± 0.03 μM) while L-AMP act as a tight-binding competitive inhibitor (K_i = 3.8 ± 0.7 nM). The K_m values obtained both for oxyluciferin and L-AMP were 14.7 ± 0.7 and 14.9 ± 0.2 μM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.