Lipoic acid was found to inhibit the firefly luciferin-luciferase reaction. The inhibition is competitive and is the strongest known (K-i = 0.026 +/- 0.013 muM) compared with other reported inhibitors. Considering the structure-activity correlations, the mechanism of inhibition may originate from the sulfur atom and carboxyl moiety of lipoic acid giving it structural specificity. Subsequent addition of lipoic acid and nitric oxide accelerated the inhibition in vitro, suggesting that lipoic acid may have a functional role in regulating firefly bioluminescence. (C) 2004 Elsevier Inc. All rights reserved.