Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A₂ (TXA₂) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA₂ in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B₂ (TXB₂), the stable metabolite of TXA₂. Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB₂ in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 ± 803 vs. 10,210 ± 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB₂ release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA₂ release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA₂ in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA₂, which is associated with upregulation of the COX-2 enzyme.