The hepatobiliary disposition of rhodamine 123 (RH‐123) and its glucuronidated (RH‐Glu) and deacylated (RH‐110) metabolites were studied in an isolated perfused rat liver (IPRL) model in the presence and absence of P‐glycoprotein (P‐gp) and Mrp2 inhibitors. A single dose (180 µg) of RH‐123 was added to a recirculating perfusate in the absence (Control) or presence of cyclosporine A (CyA) or dibromosulfophthalein (DBSP) in the perfusate. Serial (0–90 min) perfusate and bile and terminal liver samples were collected for analysis by HPLC. In the Control livers, 25.4 ± 2.2% (mean ± SD) of the dose was recovered as RH‐123 (11.7 ± 2.0%) and RH‐Glu (13.2 ± 0.9%) in the bile. Whereas CyA substantially (90%) reduced (p < 0.001) the biliary excretion of RH‐123 without affecting the excretion of RH‐Glu, DBSP reduced the biliary excretion of RH‐Glu by >80% (p < 0.001) with no effect on the biliary excretion of RH‐123. Mass balance studies showed that DBSP, in addition to reducing the biliary clearance of RH‐Glu, also strongly inhibited the glucuronidation of RH‐123, an effect that was confirmed in vitro using the glucuronidation marker umbelliferone. It is concluded that the use of RH‐123 in an IPRL model may serve as a dual marker for the determination of the altered functions of P‐gp and/or Mrp2. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:455–466, 2010