Ethyl sulfate, a minor and direct ethanol metabolite in adult human body, has been implicated as a biomarker for alcohol consumption and in utero exposure to ethanol. To understand better the physiological relevance of the sulfation of ethanol, it is important to clarify the cytosolic sulfotransferase (SULT) enzymes that are responsible for ethanol sulfation. The present study aimed to identify the major ethanol-sulfating human SULTs and to investigate the sulfation of ethanol under the metabolic setting. A systematic analysis revealed four ethanol-sulfating SULTs, SULT1A1, SULT1A2, SULT1A3, and SULT1C4, among the eleven human SULT enzymes previously prepared and purified. A metabolic labeling study demonstrated the generation and release of ethyl [(35)S]sulfate in a concentration-dependent manner by HepG2 human hepatoma cells labeled with [(35)S]sulfate in the presence of different concentrations of ethanol. Cytosol or S9 fractions of human lung, liver, and small intestine were examined to verify the presence of ethanol-sulfating activity in vivo. Of the three human organs, the small intestine displayed the highest activity.