The site of formation of 1-thiocarbamoyl-2-imidazolidinone (TCI), a potent immunoactive metabolite of the antihelminthic drug [used to treat human Schistosoma-mansoni infections], niridazole [1-(nitro-2-thiazoyl)-2-imidazolidinone]. When niridazole was administered intragastrically to C57Bl/6J mice, a 4-h delay was observed before TCI was detected in the serum. 4-Hydroxyniridazole, a marker of hepatic niridazole metabolism, appeared in the serum within 30 min. Changing the route of niridazole administration from intragastric to intracecal abolished the lag period in the rise of serum TCI concentrations relative to the 4-hydroxyniridazole marker. Pretreatment of mice with neomycin sulfate reduced the amount of TCI excreted in the urine by .apprx. 90% over a 24 h period, but did not affect the amount of 4-hydroxyniridazole excreted. Injection of niridazole into isolated segments of mouse intestine caused TCI production, with the greatest conversion noted in the cecum. Subsequent incubation of niridazole with suspensions of mouse cecum contents in vitro caused the formation of TCI, but not 4-hydroxyniridazole. Attempts to demonstrate TCI formation in vitro with various fractions of mouse liver were unsuccessful. TCI formation is not associated with the major hepatic pathway of niridazole metabolism. TCI may be formed from niridazole in the gastrointestinal tract due to the action of intestinal microflora.