Hydrogen polysulfides (H 2S n) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H 2S), which has various physiological roles. We recently found H 2S n in the brain. H 2S n induced some responses previously attributed to H 2S but with much greater potency than H 2S. However, the number of sulfur atoms in H 2S n and its producing enzyme were unknown. Here, we detected H 2S 3 and H 2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H 2S 3 and H 2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H 2S 3 from 3 MP, while those expressing defective 3MST mutants did not. H 2S 3 was localized in the cytosol of cells. H 2S 3 was also produced from H 2S by 3MST and rhodanese. H 2S 2 was identified as a minor H 2S n, and 3 MP did not affect the H 2S 5 level. The present study provides new insights into the physiology of H 2S 3 and H 2S, as well as novel therapeutic targets for diseases in which these molecules are involved.