Colonic bacteria produce hydrogen sulfide, a toxic compound postulated to play a pathogenetic role in ulcerative colitis. Colonic sulfide exposure has previously been assessed via measurements of fecal sulfide concentration. However, we found that <1% of fecal sulfide of rats was free, the remainder being bound in soluble and insoluble complexes. Thus fecal sulfide concentrations may reflect sulfide binding capacity rather than the toxic potential of feces. We utilized bismuth subnitrate to quantitate intracolonic sulfide release based on observations that bismuth 1) avidly binds sulfide; 2) quantitatively releases bound sulfide when acidified; and 3) does not influence fecal sulfide production by fecal homogenates. Rats ingesting bismuth subnitrate excreted 350 +/- 18 micromol/day of fecal sulfide compared with 9 +/- 1 micromol/day in control rats. Thus the colon normally absorbs approximately 340 micromol of sulfide daily, a quantity that would produce local and systemic injury if not efficiently detoxified by the colonic mucosa. Studies utilizing bismuth should help to clarify the factors influencing sulfide production in the human colon.