Incorporation of 18O into the CO2 produced in the bioluminescent oxidation of firefly luciferin was studied. In H216O medium with 18O2 gas, the product CO2 contained up to 75% C16O18O, showing that one O of the product CO2 arose from the O2 that oxidized luciferin. This result is consistent with a dioxetane mechanism. Analysis of the mass spectral data of the CO2 obtained in high-enrichment H218O medium with 16O2 gas indicated the presence of about 20% contaminating CO2, which contributes approximately 70% of the total incorporated 18O. Thus the values of incorporated 18O in H218O medium with 16O2 gas have no significance in the present context. Data obtained with luciferases of the American firefly Photinus and Japanese firefly Luciola were similar.