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C olonic bacteria liberate large quantities of the highly toxic gases hydrogen sulfide (H₂S) and methanethiol (CH₃SH). The colonic mucosa presumably has an efficient means of detoxifying these compounds, which is thought to occur through methylation of H₂S to CH₃SH and CH₃SH to dimethylsulfide (CH₃SCH₃). We investigated this detoxification pathway by incubating rat cecal mucosal homogenates with gas containing H₂S, CH₃SH, or CH₃SCH₃. Neither CH₃SH nor CH₃SCH₃ was produced during H₂S catabolism, whereas catabolism of CH₃SH liberated H₂S but not CH₃SCH₃. Thus, H₂S and CH₃SH are not detoxified by methylation to CH₃SCH₃. Rather, CH₃SH is demethylated to H₂S, and H₂S is converted to nonvolatile metabolites. HPLC analysis of the homogenate showed the metabolite to be primarily thiosulfate. Analysis of cecal venous blood obtained after intracecal instillation of H₂³⁵S revealed that virtually all absorbed H₂S had been oxidized to thiosulfate. The oxidation rate of H₂S by colonic mucosa was 10,000 times greater than the reported methylation rate. Conversion to thiosulfate appears to be the mechanism whereby the cecal mucosa protects itself from the injurious effects of H₂S and CH₃SH, and defects in this detoxification possibly could play a role in colonic diseases such as ulcerative colitis.