Sulfotransferases (STs) catalyze the sulfation of many structurally diverse molecules. Enzymatic assays and Western blots have been used to identify and characterize STs in the human gastrointestinal tract. Sulfation activities for 2-naphthol, dopamine, estradiol, and dehydroepiandrosterone (DHEA) from 23 donors were measured in cytosol prepared from stomach, duodenum, segments of small intestine, and colon and were compared to levels in human liver cytosol. Stomach and colon had low 2-naphthol and dopamine sulfation activities and almost no estradiol and DHEA sulfation activity. For all four substrates, small intestine has higher activities than both stomach and colon. Human small intestine 2-naphthol sulfation specific activity is approximately half that of human liver. Human small intestine dopamine sulfation activity is three times as high as that of human liver. While estrogen sulfation activity is about the same for both human intestine and human liver, human liver DHEA sulfation activity is about five times as high as that of human small intestine. The distribution of ST activities along the length of the small intestine was very different among different donors. Some donors had higher activity in the proximal segments of the small intestine, whereas other donors had higher activity in the distal segments of the small intestine. Our results also demonstrated high variation of small intestine sulfation activities compared with human liver activities among different donors. The Western blot results agreed with the enzymatic assay results. These results suggest that xenobiotics may regulate human small intestinal STs.