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Lactobacillus reuteri is a prominent member of the Lactobacillus population in the gastrointestinal ecosystem of humans, poultry, swine, and other animals. Reuterin is a newly discovered, broad-spectrum antimicrobial substance produced by this species during fermentation of glycerol. In this report, we describe procedures for (i) producing reuterin in sufficient amounts to isolate from a fermentation mixture and (ii) isolating this substance by high-performance liquid chromatography. By using uniformly labeled [14C]glycerol, reuterin was identified as a product of glycerol fermentation associated with the production of beta-hydroxypropionic acid and trimethylene glycol.