We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC–MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5–6.8 μmol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28–95 μmol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium . Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo , although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo .