H2S is highly toxic and selectively inhibits butyrate oxidation in colonocytes. Ineffective detoxification may result in mucosal insult, inflammation, and ultimately in colorectal cancer (CRC). Rhodanese can detoxify H2S and is comprised of two isoenzymes: thiosulfate sulfurtransferase (TST) and mercaptopyruvate sulfurtransferase (MST). Using specific antisera to discriminate TST from MST, we found that only TST could detoxify H2S. In sections of normal colon, both enzymes were located on the luminal mucosal surface, and they were expressed in the colonocytes but not in the mucin-secreting goblet cells. Expression of both enzymes was focally lost in ulcerative colitis and markedly reduced in advanced colon cancer, the disease progression correlating with the decreased expression of MST and TST. In HT-29 cells, a human colon cancer cell line, TST activity and expression were significantly increased by butyrate and by histone deacetylase inhibition, agents that promote HT-29 cell differentiation. Sulfide (0.1 mM) also increased TST activity, but higher sulfide concentrations (0.3-3 mM) were toxic. Preincubation in butyrate to increase TST expression, decreased sensitivity of the cells to sulfide toxicity. We conclude that decreased expression of TST (or MST) is a tumor marker for CRC. TST expression is increased in colonocyte differentiation. Dysregulation of TST expression and activity resulting in inability to effectively detoxify could be a factor in the cell loss and inflammation that accompany ulcerative colitis and ultimately then in CRC.