Glutathione (GSH) plays an important role in the metabolism of arsenite and arsenate by generating arsenic-glutathione complexes. Although dimethylarsinic acid (DMA V) is the major metabolite of inorganic arsenicals (iAs) in urine, it is not clear how DMA V is produced from iAs. In the present study we report that S-(dimethylarsino)-glutathione (DMA III(SG)), a putative precursor of dimethylarsinic acid DMA V, was unstable in the culture medium without excess GSH and generated volatile substances which were highly cytotoxic for both rat heart microvascular endothelial cells and HL60, a human leukemia cell line. Cytotoxicity of DMA III(SG) was higher than that of iAs and its LC 50 value was calculated to be 7.8 μM in the endothelial cells. To our surprise DMA III(SG) effectively killed cells in the neighbor wells of the same multi-well dish, indicating that volatile toxic compounds generated from DMA III(SG) in the culture medium. High performance lipid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICPMS) analyses suggested that the freshly generated volatile compounds dissolved into aqueous solution and formed an unstable arsenic compound and the unstable compound was further converted to DMA V. These results suggested that DMA III(SG) exerts its cytotoxicity by generating volatile arsenicals and is implicated in the metabolic conversion of inorganic arsenicals into DMA V, a major final metabolite of inorganic arsenicals in most mammals.