The enterohepatic circulation of T-2 toxin and its conjugated metabolites was examined in bile duct-cannulated male rats. Rats administered tritiated T-2 toxin intraduodenally (id) eliminated 44.65% and 57.25% of the administered dose in the bile within 4 and 8 hr post-dosing, respectively. TLC profiles of the T-2 metabolites were similar after intravascular and id administration. The major metabolites detected were 3'-OH-hydroxytryptamine-2 (HT-2), glucuronic acid conjugates, T-2 tetraol (TOL), 4-deacetylneosolaniol (4-DN), and HT-2. Tritium-labeled glucuronides obtained from the bile of rats administered [3H]T-2 toxin intravascularly were extracted and purified using C-18 and silica column chromatography. Enzymatic hydrolysis followed by TLC and GC/MS indicated that the aglycone portion of the glucuronides were composed of 3'-OH HT-2, HT-2, 4-DN, and TOL. After id administration of the glucuronides the rats eliminated 6.01% (4 hr) and 11.86% (8 hr) of the dose in the bile. No free metabolites of T-2 toxin were detected in the bile of any animals administered the purified glucuronides. Oral treatment of the rats with the beta-glucuronidase inhibitor, saccharolactone, did not produce a significant decline in the amount of radioactivity recovered in the bile following administration of the tritium-labeled glucuronides. These studies substantiate the enterohepatic circulation of T-2 toxin metabolites.