Naproxen is a nonsteroidal anti-inflammatory drug widely used as an analgesic and anti-inflammatory agent. The conjugated forms of naproxen and O-DMN, its demethylated metabolite, account for 66-92% of naproxen found in human urine. In this study, O-DMN and structurally related compounds were tested as substrates for seven isoforms of human cytosolic sulfotransferase (SULT). The SULT2 or hydroxysteroid SULT isoforms, SULT2A1 and SULT2B1b, did not show reactivity with any of the compounds. All five SULT1 isoforms were active although there was variability between SULT isoforms and compounds assayed. O-DMN was sulphated by SULT1A1, SULT1B1 and SULT1E1. All five SULT1 isoforms were capable of conjugating both α-naphthol and β-naphthol. Apparent Km values for O-DMN sulphation were significantly higher than the values for either α-naphthol or β-naphthol. SULTs 1A1, 1B1 and 1E1 had Kms for O-DMN sulphation of 84 μM, 690 μM, and 341 μM, respectively. These Km values were 40-1150-fold higher than the Km values for α- and β-naphthol. The role of the side-chain in O-DMN sulphation was studied using a series of structurally related β-naphthol compounds as substrates for SULT1A1 and SULT1E1. The presence of lipophilic groups increased affinity for both SULT isoforms whereas inclusion of a carboxyl group inhibited activity. These studies indicate that O-DMN is sulphated by SULT1A1, B1 and 1E1. Because of the high concentrations of SULT1A1 expression in human liver and intestines and its higher affinity for O-DMN sulphation, SULT1A1 may have a role in the first pass metabolism of O-DMN. © 2005 Blackwell Publishing Ltd.