Hydrogen sulfide (H₂S) is endogenously produced from L-cysteine (L-Cys) by cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The third enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) produces H₂S from 3-mercaptopyruvate (3-MP), which is provided from L-Cys and α-ketoglutarate (α-KG) by cysteine aminotransferase (CAT). Recent studies have shown that H₂S promotes growth and proliferation of tumor cells. H₂S is expected to provide a new perspective in elucidating the pathology of malignant tumors; however, the production of H₂S in glioblastoma, the most aggressive and malignant type of astrocytoma, is largely unknown. In this study, we examined the production of H₂S in human glioblastoma cell line U-251 MG. Cell lysates of U-251 MG produced H₂S from 3-MP, and the production of H₂S was inhibited by a selective inhibitor of 3-MST HMPSNE. Because 3-MP is provided by CAT from L-Cys and α-KG, we then examined the possibility that 3-MST with CAT produces H₂S. The lysates produced H₂S from L-Cys in the presence of α-KG. In the absence of α-KG H₂S from L-Cys markedly reduced, suggesting that H₂S production is highly dependent on the activity of CAT. This conclusion is supported by the observation that H₂S production from L-Cys and a-KG was suppressed by L-aspartate, a substrate with higher affinity than L-Cys. These results suggest that 3-MST with CAT may function as the major H₂S-producing enzyme in glioblastoma cells.