Thiothiamine was intraperitoneally injected in rats and the urinary metabolites were investigated by paper chromatography using acetic acid-n-butanol-water and 3 spots at RF 0.88-0.95, 0.7 and 0.3 were detected. The spot at RF 0.7 corresponded to thiothiamine. The alkalinized urine was extracted with ether, then with isobutanoL Thiothiamine was obtained from the ether phase. The isobutanol layer was evaporated and the residue was subjected to cellulose column chromatography to yield oily substances of RF 0.88 and 0.95. Further purification by paper chromatography led to the isolation of a compound positive for sulfhydryl group with a maximum at 320 m/i in ultraviolet absorption spectrum. It was identified as 2-mercapto-4-methyl-5-[beta]-hydroxyethylthiazole by elementary analysis, mixed melting point and paper chromatography. Further confirmation was made with its picrate.