Buprenorphine, pentazocine, and naloxone are opioid drugs used for the treatment of pain and opioid dependence or overdose. Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is involved in the metabolism of a variety of xenobiotics including drug compounds. Sulfation of opioid drugs has not been well investigated. The current study was designed to examine the sulfation of three opioid drugs, buprenorphine, pentazocine, and naloxone, in HepG2 human hepatoma cells and to identify the human SULT(s) responsible for their sulfation. Analysis of the spent media of HepG2 cells, metabolically labeled with [35S]sulfate in the presence of each of the three opioid drugs, showed the generation and release of their [35S]sulfated derivatives. A systematic analysis using eleven known human SULTs revealed SULT1A3 and SULT2A1 as the major responsible SULTs for the sulfation of, respectively, pentazocine and buprenorphine; whereas three other SULTs, SULT1A1, SULT1A2, and SULT1C4, were capable of sulfating naloxone. Enzymatic assays using combinations of these opioid drugs as substrates showed significant inhibitory effects in the sulfation of buprenorphine and pentazocine by naloxone. Differential sulfating activities toward the three opioid drugs were detected in cytosol or S9 fractions of human lung, liver, kidney, and small intestine. Collectively, these results imply that sulfation may play a role in the metabolism of buprenorphine, pentazocine, and naloxone in vivo.