The bioluminescent reaction of Oplophorus takes place when the oxidation of coelenterazine (the luciferin) with molecular oxygen is catalyzed by Oplophorus luciferase, resulting in light of maximum intensity at 462 nm and the products CO2 and coelenteramide. Oplophorus luciferase has now been obtained in a highly purified state. Optimum luminescence occurs at pH 9 in the presence of 0.05--0.1 M NaCl at 40 degrees C, and, due to the unusual resistance of this enzyme to heat, visible luminescence occurs at temperatures above 70 degrees C when partially purified enzyme is used. The specific activity of purest preparations is 1.75 X 10(15) photons s-1 mg-1 at 23 degrees C. At pH 8.7, native luciferase has a molecular weight of approximately 130 000, apparently comprising 4 monomers of 31 000; at lower pHs, the native luciferase tends to polymerize. The quantum yield of coelenterazine is 0.34 at 22 degrees C with this enzyme. After the luminescent reaction, the spent solution is nonfluorescent, and likewise solutions of luciferase alone. When the bioluminescent reaction was carried out in the presence of 18O2, the product CO2 contained more than 50% C18O16O, supporting the dioxetane mechanism, but without ruling out the linear peroxide mechanism.