1. The activities of the microsomal thiol methyltransferase and the cytosolic thiopurine methyltransferase were measured with 2-mercaptoethanol and 6-mercaptopurine as substrates in human ileum, ascending colon, transverse colon, descending colon and liver. 2. Thiol methyltransferase activity (pmol/min per mg) (mean +/- SD) was 495 +/- 280 (ileum), 786 +/- 454 (ascending colon), 1791 +/- 233 (transverse colon), 964 +/- 484 (descending colon) and 4800 +/- 1194 (liver). 3. Thiopurine methyltransferase (pmol/min per mg) (mean +/- SD) was 53.5 +/- 15.4 (ileum), 34.6 +/- 11.4 (ascending colon), 64.3 +/- 12.1 (transverse colon), 57.0 +/- 10.1 (descending colon) and 106 +/- 20.4 (liver). 4. Transferase in intestinal mucosa followed non-Michaelis-Menten kinetics, and two phases representing high and low affinity forms, for the acceptor methyl substrates were observed. 5. Comparison of intestinal with hepatic activities showed that thiopurine methyltransferase is better expressed than thiol methyltransferase in the human intestine, at least with the substrates studied.