The acute 2-hour effects of isoflurane anesthesia on liver function and biliary excretion were examined in 2 ponies prepared surgically with chronic external biliary fistulas (T-tubes). Studies were conducted 2 to 8 months postoperatively with the enterohepatic circulation held intact between studies. Bile acid infusion IV (8.1 to 8.8 mumol/min) helped maintain bile flow and bile acid and bilirubin excretion during complete biliary diversion throughout each study. Following 3-hour control periods, anesthesia was induced and maintained at 1.3 to 1.5 minimal alveolar concentration plus O2 (spontaneous breathing) for 2 hours. Compared with the immediate 2-hour preanesthesia values, isoflurane caused significant increases in PCV (27%) and biliary bilirubin excretion (24%). However, no significant differences were detected in plasma or biliary bilirubin concentrations, biliary bile acid concentration or excretion, bile flow, or plasma aspartate aminotransferase concentrations between preanesthesia control and anesthesia periods. The results indicate that although isoflurane anesthesia enhanced hepatic bilirubin excretion, its effects on hepatic bilirubin formation and/or clearance are modest, compared with effects of halothane anesthesia which have previously been shown to enhance equine bilirubin excretion by 138% and reduce bile acid excretion by 27%. Isoflurane anesthesia in ponies does not appear to affect hepatic bile acid transport or bile formation significantly.