The role of biliary elimination in the metabolic disposition of 2,4-D was evaluated in male and female Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters. Following cannulation of the bile duct, an intragastric (ig) dose of 2,4-D (200 mg/kg) was administered and bile was collected at 30- or 60-min intervals for up to 6 h. Bile flow rates were constant in rats, increased in mice, and decreased in hamsters throughout the collection periods. Total recovery of radioactivity was greatest in male mice (about 7% of administered dose over 4 h). Female mice and rats of both sexes excreted about 3% over the same interval and male and female hamsters about 1%. About 71–88% of the activity in bile was parent compound. The glycine conjugate of 2,4-D was found in bile from mice, rats, and hamsters and the taurine conjugate in bile from mice. The only sex-dependent difference in the metabolite profile was in mice. Male mice excreted twice as much glycine conjugate as female mice. An additional minor metabolite (4–7%) was present in rat and mouse bile. This was tentatively identified as 2,4-D-glucuronide based on its hydrolysis by β-glucuronidase. One more very minor metabolite (3%) was detected in rat bile but was not characterized due to its lability. The results of this study indicate that there are species-dependent differences in the biliary elimination of 2,4-D but not sex-dependent differences.