Hydrogen sulfide (H₂S) has been recognized as a smooth muscle relaxant. Cystathionine γ-lyase, which is localized to smooth muscle, is thought to be the major H₂S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H₂S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H₂S from cysteine and α-ketoglutarate. The present study provides a new insight into the production and release of H₂S as a smooth muscle relaxant from vascular endothelium.